Axon Initial Segment Research Articles

Effect of Vectashield-induced fluorescence quenching on conventional and super-resolution microscopy

Finding the right combination of a fluorescent dye and a mounting medium is crucial for optimal microscopy of fixed samples. It was recently shown that Vectashield, one of the most commonly used mounting media for conventional microscopy, can also be applied to super-resolution direct stochastic optical reconstruction microscopy (dSTORM). dSTORM utilizes conventional dyes and starts with samples in a fluorescent “ON” state. This helps in identifying structures of interest. Subsequently, labelled samples are induced into blinking, which is necessary for determining the position of single molecules and reconstruction of super-resolution images. This is only possible with certain fluorescent dyes and imaging buffers. One of the most widely used dyes for dSTORM, Alexa Fluor 647 (AF647), blinks in Vectashield. However, after preparing immunocytochemical samples in Vectashield, we noticed that the fluorescence intensity of AF647 is quenched. This is particularly evident for dimmer immunostainings, such as stainings of some components of neuronal cytoskeleton and axonal initial segment. Because structures of interest cannot be identified in quenched samples, loss of fluorescence intensity hinders imaging of AF647 in Vectashield. This has consequences for both conventional and dSTORM imaging. To overcome this, we provide: 1) a quantitative analysis of AF647 intensity in different imaging media, 2) a quantitative analysis of the suitability of Vectashield for dSTORM imaging of high and low-abundance AF647-labelled targets. Furthermore, for the first time, we quantitatively analyse the performance of Alexa Fluor Plus 647, a new variant of AF647-conjugated antibody, in dSTORM imaging.

Tropomyosin Tpm3.1 Is Required to Maintain the Structure and Function of the Axon Initial Segment.

SummaryThe axon initial segment (AIS) is the site of action potential initiation and serves as a cargo transport filter and diffusion barrier that helps maintain neuronal polarity. The AIS actin cytoskeleton comprises actin patches and periodic sub-membranous actin rings. We demonstrate that tropomyosin isoform Tpm3.1 co-localizes with actin patches and that the inhibition of Tpm3.1 led to a reduction in the density of actin patches. Furthermore, Tpm3.1 showed a periodic distribution similar to sub-membranous actin rings but Tpm3.1 was only partially congruent with sub-membranous actin rings. Nevertheless, the inhibition of Tpm3.1 affected the uniformity of the periodicity of actin rings. Furthermore, Tpm3.1 inhibition led to reduced accumulation of AIS structural and functional proteins, disruption in sorting somatodendritic and axonal proteins, and a reduction in firing frequency. These results show that Tpm3.1 is necessary for the structural and functional maintenance of the AIS.

M-Current Inhibition in Hippocampal Excitatory Neurons Triggers Intrinsic and Synaptic Homeostatic Responses at Different Temporal Scales.

Persistent alterations in neuronal activity elicit homeostatic plastic changes in synaptic transmission and/or intrinsic excitability. However, it is unknown whether these homeostatic processes operate in concert or at different temporal scales to maintain network activity around a set-point value. Here we show that chronic neuronal hyperactivity, induced by M-channel inhibition, triggered intrinsic and synaptic homeostatic plasticity at different timescales in cultured hippocampal pyramidal neurons from mice of either sex. Homeostatic changes of intrinsic excitability occurred at a fast timescale (1-4 h) and depended on ongoing spiking activity. This fast intrinsic adaptation included plastic changes in the threshold current and a distal relocation of FGF14, a protein physically bridging Nav1.6 and Kv7.2 channels along the axon initial segment. In contrast, synaptic adaptations occurred at a slower timescale (∼2 d) and involved decreases in miniature EPSC amplitude. To examine how these temporally distinct homeostatic responses influenced hippocampal network activity, we quantified the rate of spontaneous spiking measured by multielectrode arrays at extended timescales. M-Channel blockade triggered slow homeostatic renormalization of the mean firing rate (MFR), concomitantly accompanied by a slow synaptic adaptation. Thus, the fast intrinsic adaptation of excitatory neurons is not sufficient to account for the homeostatic normalization of the MFR. In striking contrast, homeostatic adaptations of intrinsic excitability and spontaneous MFR failed in hippocampal GABAergic inhibitory neurons, which remained hyperexcitable following chronic M-channel blockage. Our results indicate that a single perturbation such as M-channel inhibition triggers multiple homeostatic mechanisms that operate at different timescales to maintain network mean firing rate.SIGNIFICANCE STATEMENT Persistent alterations in synaptic input elicit homeostatic plastic changes in neuronal activity. Here we show that chronic neuronal hyperexcitability, induced by M-type potassium channel inhibition, triggered intrinsic and synaptic homeostatic plasticity at different timescales in hippocampal excitatory neurons. The data indicate that the fast adaptation of intrinsic excitability depends on ongoing spiking activity but is not sufficient to provide homeostasis of the mean firing rate. Our results show that a single perturbation such as M-channel inhibition can trigger multiple homeostatic processes that operate at different timescales to maintain network mean firing rate.

Fractal, Scale Free Electromagnetic Resonance of a Single Brain Extracted Microtubule Nanowire, a Single Tubulin Protein and a Single Neuron

Biomaterials are primarily insulators. For nearly a century, electromagnetic resonance and antenna–receiver properties have been measured and extensively theoretically modeled. The dielectric constituents of biomaterials—if arranged in distinct symmetries, then each vibrational symmetry—would lead to a distinct resonance frequency. While the literature is rich with data on the dielectric resonance of proteins, scale-free relationships of vibrational modes are scarce. Here, we report a self-similar triplet of triplet resonance frequency pattern for the four-4 nm-wide tubulin protein, for the 25-nm-wide microtubule nanowire and 1-μm-wide axon initial segment of a neuron. Thus, preserving the symmetry of vibrations was a fundamental integration feature of the three materials. There was no self-similarity in the physical appearance: the size varied by 106 orders, yet, when they vibrated, the ratios of the frequencies changed in such a way that each of the three resonance frequency bands held three more bands inside (triplet of triplet). This suggests that instead of symmetry, self-similarity lies in the principles of symmetry-breaking. This is why three elements, a protein, it’s complex and neuron resonated in 106 orders of different time domains, yet their vibrational frequencies grouped similarly. Our work supports already-existing hypotheses for the scale-free information integration in the brain from molecular scale to the cognition.

Theoretical relation between axon initial segment geometry and excitability.

In most vertebrate neurons, action potentials are triggered at the distal end of the axon initial segment (AIS). Both position and length of the AIS vary across and within neuron types, with activity, development and pathology. What is the impact of AIS geometry on excitability? Direct empirical assessment has proven difficult because of the many potential confounding factors. Here, we carried a principled theoretical analysis to answer this question. We provide a simple formula relating AIS geometry and sodium conductance density to the somatic voltage threshold. A distal shift of the AIS normally produces a (modest) increase in excitability, but we explain how this pattern can reverse if a hyperpolarizing current is present at the AIS, due to resistive coupling with the soma. This work provides a theoretical tool to assess the significance of structural AIS plasticity for electrical function.

Held in the balance.

Chandelier cells, a type of cortical interneuron targeting the axon initial segment of pyramidal cells are shown to monitor network activity and adjust their output in a homeostatic manner.

The FTLD Risk Factor TMEM106B Regulates the Transport of Lysosomes at the Axon Initial Segment of Motoneurons

Genetic variations in TMEM106B, coding for a lysosomal membrane protein, affect frontotemporal lobar degeneration (FTLD) in GRN- (coding for progranulin) and C9orf72-expansion carriers and might play a role in aging. To determine the physiological function of TMEM106B, we generated TMEM106B-deficient mice. These mice develop proximal axonal swellings caused by drastically enlarged LAMP1-positive vacuoles, increased retrograde axonal transport of lysosomes, and accumulation of lipofuscin and autophagosomes. Giant vacuoles specifically accumulate at the distal end and within the axon initial segment, but not in peripheral nerves or at axon terminals, resulting in an impaired facial-nerve-dependent motor performance. These data implicate TMEM106B in mediating the axonal transport of LAMP1-positive organelles in motoneurons and axonal sorting at the initial segment. Our data provide mechanistic insight into how TMEM106B affects lysosomal proteolysis and degradative capacity in neurons.

Activity-Dependent Plasticity of Axo-axonic Synapses at the Axon Initial Segment

SummaryThe activity-dependent rules that govern the wiring of GABAergic interneurons are not well understood. Chandelier cells (ChCs) are a type of GABAergic interneuron that control pyramidal cell output through axo-axonic synapses that target the axon initial segment. In vivo imaging of ChCs during development uncovered a narrow window (P12–P18) over which axons arborized and formed connections. We found that increases in the activity of either pyramidal cells or individual ChCs during this temporal window result in a reversible decrease in axo-axonic connections. Voltage imaging of GABAergic transmission at the axon initial segment (AIS) showed that axo-axonic synapses were depolarizing during this period. Identical manipulations of network activity in older mice (P40–P46), when ChC synapses are inhibitory, resulted instead in an increase in axo-axonic synapses. We propose that the direction of ChC synaptic plasticity follows homeostatic rules that depend on the polarity of axo-axonic synapses.

Nodal β spectrins are required to maintain Na+ channel clustering and axon integrity.

Clustered ion channels at nodes of Ranvier are critical for fast action potential propagation in myelinated axons. Axon-glia interactions converge on ankyrin and spectrin cytoskeletal proteins to cluster nodal Na+ channels during development. However, how nodal ion channel clusters are maintained is poorly understood. Here, we generated mice lacking nodal spectrins in peripheral sensory neurons to uncouple their nodal functions from their axon initial segment functions. We demonstrate a hierarchy of nodal spectrins, where β4 spectrin is the primary spectrin and β1 spectrin can substitute; each is sufficient for proper node organization. Remarkably, mice lacking nodal β spectrins have normal nodal Na+ channel clustering during development, but progressively lose Na+ channels with increasing age. Loss of nodal spectrins is accompanied by an axon injury response and axon deformation. Thus, nodal spectrins are required to maintain nodal Na+ channel clusters and the structural integrity of axons.

Multiple Domains in the Kv7.3 C-Terminus Can Regulate Localization to the Axon Initial Segment.

The voltage-gated Kv7.2/Kv7.3 potassium channel is a critical regulator of neuronal excitability. It is strategically positioned at the axon initial segment (AIS) of neurons, where it effectively inhibits repetitive action potential firing. While the selective accumulation of Kv7.2/Kv7.3 channels at the AIS requires binding to the adaptor protein ankyrin G, it is currently unknown if additional molecular mechanisms contribute to the localization and fine-tuning of channel numbers at the AIS. Here, we utilized a chimeric approach to pinpoint regions within the Kv7.3 C-terminal tail with an impact upon AIS localization. This strategy identified two domains with opposing effects upon the AIS localization of Kv7.3 chimeras expressed in cultured hippocampal neurons. While a membrane proximal domain reduced AIS localization of Kv7.3 chimeras, helix D increased and stabilized chimera AIS localization. None of the identified domains were required for AIS localization. However, the domains modulated the relative efficiency of the localization raising the possibility that the two domains contribute to the regulation of Kv7 channel numbers and nanoscale organization at the AIS.

Calcineurin Signaling Mediates Disruption of the Axon Initial Segment Cytoskeleton after Injury.

SummaryThe axon initial segment (AIS) cytoskeleton undergoes rapid and irreversible disruption prior to cell death after injury, and loss of AIS integrity can produce profound neurological effects on the nervous system. Here we described a previously unrecognized mechanism for ischemia-induced alterations in AIS integrity. We show that in hippocampal CA1 pyramidal neurons Nav1.6 mostly preserves at the AIS after disruption of the cytoskeleton in a mouse model of middle cerebral artery occlusion. Genetic removal of neurofascin-186 leads to rapid disruption of Nav1.6 following injury, indicating that neurofascin is required for Nav1.6 maintenance at the AIS after cytoskeleton collapse. Importantly, calcineurin inhibition with FK506 fully protects AIS integrity and sufficiently prevents impairments of spatial learning and memory from injury. This study provides evidence that calcineurin activation is primarily involved in initiating disassembly of the AIS cytoskeleton and that maintaining AIS integrity is crucial for therapeutic strategies to facilitate recovery from injury.

Structural Evolution of Energy-Consuming Tau Mediated Microtubule Bundles

Tau is an intrinsically disordered neuronal protein known to modulate microtubule dynamics through the suppression of dynamic instability. Recent studies have shown that Tau mediates dynamical linear microtubule bundles similar to microtubule fascicles observed in vivo, which are a cardinal feature of the axon initial segment (P. J. Chung, C. Song, et al. Nature Communications 2016, 7, 12278. DOI: 10.1038/ncomms12278 and ACS Macro Lett. 2018, 7, 228-232. DOI: 10.1021/acsmacrolett.7b00937). In order to better understand the architecture of these nanoscale energy-consuming dissipative structures, quantitative techniques of synchrotron small-angle x-ray scattering data—combined with TEM, and other analytical techniques—have been utilized. Here we show that under biologically relevant GTP and Mg2+ concentrations, microtubule bundles undergo a time induced transition, in which the wall to wall distance of the previously observed “pseudo-hexagonal” wide spacing dramatically drops, and remodels to form a tightly bound, “locked-hexagonal” structure, which in turn, induces a rapid depolymerization of the microtubule bundle. The structure and stability of these architectures appear to be Tau-isoform dependent and underscore the importance of the expression of distinct Tau-isoforms within neurons.

Bridging the Molecular-Cellular Gap in Understanding Ion Channel Clustering.

The clustering of many voltage-dependent ion channel molecules at unique neuronal membrane sites such as axon initial segments, nodes of Ranvier, or the post-synaptic density, is an active process mediated by the interaction of ion channels with scaffold proteins and is of immense importance for electrical signaling. Growing evidence indicates that the density of ion channels at such membrane sites may affect action potential conduction properties and synaptic transmission. However, despite the emerging importance of ion channel density for electrical signaling, how ion channel-scaffold protein molecular interactions lead to cellular ion channel clustering, and how this process is regulated are largely unknown. In this review, we emphasize that voltage-dependent ion channel density at native clustering sites not only affects the density of ionic current fluxes but may also affect the conduction properties of the channel and/or the physical properties of the membrane at such locations, all changes that are expected to affect action potential conduction properties. Using the concrete example of the prototypical Shaker voltage-activated potassium channel (Kv) protein, we demonstrate how insight into the regulation of cellular ion channel clustering can be obtained when the molecular mechanism of ion channel-scaffold protein interaction is known. Our review emphasizes that such mechanistic knowledge is essential, and when combined with super-resolution imaging microscopy, can serve to bridge the molecular-cellular gap in understanding the regulation of ion channel clustering. Pressing questions, challenges and future directions in addressing ion channel clustering and its regulation are discussed.

Profiling parvalbumin interneurons using iPSC: challenges and perspectives for Autism Spectrum Disorder (ASD)

Autism spectrum disorders (ASD) are persistent conditions resulting from disrupted/altered neurodevelopment. ASD multifactorial etiology—and its numerous comorbid conditions—heightens the difficulty in identifying its underlying causes, thus obstructing the development of effective therapies. Increasing evidence from both animal and human studies suggests an altered functioning of the parvalbumin (PV)-expressing inhibitory interneurons as a common and possibly unifying pathway for some forms of ASD. PV-expressing interneurons (short: PVALB neurons) are critically implicated in the regulation of cortical networks’ activity. Their particular connectivity patterns, i.e., their preferential targeting of perisomatic regions and axon initial segments of pyramidal cells, as well as their reciprocal connections, enable PVALB neurons to exert a fine-tuned control of, e.g., spike timing, resulting in the generation and modulation of rhythms in the gamma range, which are important for sensory perception and attention.New methodologies such as induced pluripotent stem cells (iPSC) and genome-editing techniques (CRISPR/Cas9) have proven to be valuable tools to get mechanistic insight in neurodevelopmental and/or neurodegenerative and neuropsychiatric diseases. Such technological advances have enabled the generation of PVALB neurons from iPSC. Tagging of these neurons would allow following their fate during the development, from precursor cells to differentiated (and functional) PVALB neurons. Also, it would enable a better understanding of PVALB neuron function, using either iPSC from healthy donors or ASD patients with known mutations in ASD risk genes. In this concept paper, the strategies hopefully leading to a better understanding of PVALB neuron function(s) are briefly discussed. We envision that such an iPSC-based approach combined with emerging (genetic) technologies may offer the opportunity to investigate in detail the role of PVALB neurons and PV during “neurodevelopment ex vivo.”

Shorter axon initial segments do not cause repetitive firing impairments in the adult presymptomatic G127X SOD-1 Amyotrophic Lateral Sclerosis mouse

Increases in axonal sodium currents in peripheral nerves are some of the earliest excitability changes observed in Amyotrophic Lateral Sclerosis (ALS) patients. Nothing is known, however, about axonal sodium channels more proximally, particularly at the action potential initiating region - the axon initial segment (AIS). Immunohistochemistry for Nav1.6 sodium channels was used to investigate parameters of AISs of spinal motoneurones in the G127X SOD1 mouse model of ALS in adult mice at presymptomatic time points (~190 days old). In vivo intracellular recordings from lumbar spinal motoneurones were used to determine the consequences of any AIS changes. AISs of both alpha and gamma motoneurones were found to be significantly shorter (by 6.6% and 11.8% respectively) in G127X mice as well as being wider by 9.8% (alpha motoneurones). Measurements from 20–23 day old mice confirmed that this represented a change during adulthood. Intracellular recordings from motoneurones in presymptomatic adult mice, however, revealed no differences in individual action potentials or the cells ability to initiate repetitive action potentials. To conclude, despite changes in AIS geometry, no evidence was found for reduced excitability within the functional working range of firing frequencies of motoneurones in this model of ALS.

Intramuscular Botulinum toxin A injections induce central changes to axon initial segments and cholinergic boutons on spinal motoneurones in rats

Intramuscular injections of botulinum toxin block pre-synaptic cholinergic release at neuromuscular junctions producing a temporary paralysis of affected motor units. There is increasing evidence, however, that the effects are not restricted to the periphery and can alter the central excitability of the motoneurones at the spinal level. This includes increases in input resistance, decreases in rheobase currents for action potentials and prolongations of the post-spike after-hyperpolarization. The aim of our experiments was to investigate possible anatomical explanations for these changes. Unilateral injections of Botulinum toxin A mixed with a tracer were made into the gastrocnemius muscle of adult rats and contralateral tracer only injections provided controls. Immunohistochemistry for Ankyrin G and the vesicular acetylcholine transporter labelled axon initial segments and cholinergic C-boutons on traced motoneurones at 2 weeks post-injection. Soma size was not affected by the toxin; however, axon initial segments were 5.1% longer and 13.6% further from the soma which could explain reductions in rheobase. Finally, there was a reduction in surface area (18.6%) and volume (12.8%) but not frequency of C-boutons on treated motoneurones potentially explaining prolongations of the after-hyperpolarization. Botulinum Toxin A therefore affects central anatomical structures controlling or modulating motoneurone excitability explaining previously observed excitability changes.

NuMA1 facilitates the assembly of the axon initial segment by promoting the retention of neurofascin-186.

Axon initial segment (AIS) functionality relies on a specific organization of the AIS with high enrichment of structural and functional proteins. In this issue, Torii et al. (2019. J. Cell. Biol.https://doi.org/10.1083/jcb.201907048) describe a mechanism for achieving a high density of proteins in the nascent AIS.

GABA in, garbage out: AIS-located proteasomes regulate the developmental GABA switch.

The GABA response switch from excitatory to inhibitory is a key event in neuronal maturation that depends on the regulated expression of chloride transporters NKCC1 and KCC2. In this issue, Lee et al. (2020. J. Cell. Biol.https://doi.org/10.1083/jcb.201903033) describe how degradation of NKCC1 by proteasomes immobilized at the axon initial segment (AIS) by the Ecm29 adaptor contributes to this regulation, driving the GABA switch and the positional maturation of the AIS.

The clustering of voltage-gated sodium channels in various excitable membranes.

In excitable membranes, the clustering of voltage-gated sodium channels (VGSC) serves to enhance excitability at critical sites. The two most profoundly studied sites of channel clustering are the axon initial segment, where action potentials are generated and the node of Ranvier, where action potentials propagate along myelinated axons. The clustering of VGSC is found, however, in other highly excitable sites such as axonal terminals, postsynaptic membranes of dendrites and muscle fibers, and pre-myelinated axons. In this review, different examples of axonal as well as non-axonal clustering of VGSC are discussed and the underlying mechanisms are compared. Whether the clustering of channels is intrinsically or extrinsically induced, it depends on the submembranous actin-based cytoskeleton that organizes these highly specialized membrane microdomains through specific adaptor proteins.

Ecm29-mediated proteasomal distribution modulates excitatory GABA responses in the developing brain.

Neuronal GABAergic responses switch from excitatory to inhibitory at an early postnatal period in rodents. The timing of this switch is controlled by intracellular Cl- concentrations, but factors determining local levels of cation-chloride cotransporters remain elusive. Here, we report that local abundance of the chloride importer NKCC1 and timely emergence of GABAergic inhibition are modulated by proteasome distribution, which is mediated through interactions of proteasomes with the adaptor Ecm29 and the axon initial segment (AIS) scaffold protein ankyrin G. Mechanistically, both the Ecm29 N-terminal domain and an intact AIS structure are required for transport and tethering of proteasomes in the AIS region. In mice, Ecm29 knockout (KO) in neurons increases the density of NKCC1 protein in the AIS region, a change that positively correlates with a delay in the GABAergic response switch. Phenotypically, Ecm29 KO mice showed increased firing frequency of action potentials at early postnatal ages and were hypersusceptible to chemically induced convulsive seizures. Finally, Ecm29 KO neurons exhibited accelerated AIS developmental positioning, reflecting a perturbed AIS morphological plastic response to hyperexcitability arising from proteasome inhibition, a phenotype rescued by ectopic Ecm29 expression or NKCC1 inhibition. Together, our findings support the idea that neuronal maturation requires regulation of proteasomal distribution controlled by Ecm29.