ºìÌÒÊÓƵ

The axon plasma membrane skeleton (APMS) comprises a highly organised, periodic lattice of azimuthal actin rings connected by longitudinal spectrin tetramers. Computational modeling and single-particle tracking dynamics have shown that beyond structural support, the APMS regulates both axonal mechanics and membrane dynamics. Simulations performed using a coarse-grained molecular dynamics (CGMD) model, which consists of actin particles forming rings and chains of particles representing spectrin tetramers with repeats that can unfold, demonstrate that under extension, the APMS acts as a viscoelastic tension buffer, transitioning to a perfectly plastic regime governed by the sequential unfolding of spectrin repeats and the rate-dependent dissociation of actin-spectrin junctions. Furthermore, the APMS restricts membrane protein mobility via permeable steric actin fences and spectrin-lipid interactions, inducing a deeply anisotropic protein diffusion and ultimately resulting in full protein immobilisation at the mature axon initial segment. When the Hodgkin-Huxley cable equations are integrated with the nanoscale organization of the APMS, we find that the periodic azimuthal clustering of voltage-gated sodium and potassium channels has negligible effects on action potential (AP) conduction due to spatial averaging over the AP wavelength. Instead, AP waveforms and conduction velocities are primarily determined by non-uniform axonal morphologies, such as physiological or pathological beading. Narrow inter-bead connectors act as dominant electrical bottlenecks, introducing local impedance mismatches that modulate local velocity or trigger propagation failure.

Dr George Lykotrafitis

University of Connecticut, US

Dr George Lykotrafitis

University of Connecticut, US

The process of action potential generation in the AIS is a sequence of events following membrane potential depolarisation that is initiated by microsecond-scale activation of voltage-gated Na+ channels at a triggering zone. The consequent events include bi-directional active action potential propagation and concomitant activation of Na+, K+ and Ca2+ channels, shaping the action potential waveform. We developed ultrafast voltage, Na+ and Ca2+ imaging methods to unravel this sequence of events at sub-millisecond scale in the AIS of layer-5 pyramidal neurons of the mouse somatosensory cortex. I will first present an analysis performed in neurons from wild type mice, focussing on some aspects Nav1.2 Na+ channel function. Then, I will present a comparative study from neurons in two autism-spectrum-disorder mouse models with loss-of-function mutations of the SCN2A gene coding for the Nav1.2 channel. With the support of NEURON modelling, we reconstructed the functional AIS maturation in the postnatal day P21-40 period in wild-type and mutated strains. I will show how physiological AIS maturation is disrupted, notably in divergent manner, in the two transgenic strains. Functional AIS restructuring translates into changes of the action potential waveform, and therefore of neuronal excitability, that are not simply caused by Nav1.2 loss-of-function, but also by abnormal distribution of other ion channels involved in action potential generation, in particular voltage-gated K+ channels. Overall, we provide evidence of the link between developmental disease and AIS maturation.

Dr Marco Canepari

LIPhy, CNRS-UGA, Grenoble, France

Dr Marco Canepari

LIPhy, CNRS-UGA, Grenoble, France

Marco Canepari graduated in physics at the University of Genoa in 1994 and received his PhD in biophysics from the International School for Advanced Studies in Trieste in 1999. He worked at the National Institute for Medical Research in London, at Yale University and at the University of Basel before joining the Inserm in 2010. In his career, he contributed to the development of several optical techniques for neurophysiology and applied them to many problems in fundamental and translational neuroscience. In the last seven years, he developed specific imaging approaches to investigate action potential generation in the axon initial segment and complemented this experimental analysis with computational strategies to reveal the complex interplay of ion channels underlying this process. He is also involved in several international neuroscience schools training future generation of electrophysiologists.

Prolonged changes in neuronal activity are known to induce both structural and molecular adaptations at the axon initial segment (AIS). Changes in AIS position and length as well as in the abundance of specific ion channels have been reported. However, it remains unclear how the complex molecular AIS scaffold responds globally to activity changes. To characterise the global, homeostatic AIS response, we exposed cultured rat hippocampal neurons to a classical bidirectional activity-manipulation protocol and mapped the responses of a broad panel of AIS ion channels and cell adhesion molecules, their associated scaffold proteins and the cytoskeletal network. We find that the AIS responds to prolonged activity-changes with both structural and molecular adaptations, and that these responses are not simple bidirectional adaptations but instead involve both target- and direction/manipulation specific changes. Changes in total protein expression levels could not account for all AIS-specific adaptations suggesting that multiple molecular mechanisms contribute to the AIS response. We therefore suggest that prolonged changes in neuronal activity elicit a complex, target- and modulation-specific response at the AIS that remodels both its structural and molecular architecture through multiple interacting molecular mechanisms.

Dr Hanne Borger Rasmussen

University of Copenhagen, Denmark

Dr Hanne Borger Rasmussen

University of Copenhagen, Denmark

Dr Hanne Borger Rasmussen is Associate Professor at University of Copenhagen where she leads the Neuronal Ion Channels Group. She has worked on neuronal ion channels for more than 20 years. She performed her master thesis work in the laboratory of Dr Antoine Triller in Paris and continued as a PhD student working in the laboratories of both Professor Søren-Peter Olesen in Copenhagen and Professor James Trimmer at UC Davis. Her current research focuses on ion channels of the axon initial segment. Her lab aims to understand how the complex ion channel repertoire at the axon initial segment is assembled and regulated at the molecular level.

Activity level in neuronal networks is dynamically regulated to prevent network hyper- or hypo-activity. Homeostatic plasticity mechanisms allow the fine-tuning of synaptic strength and intrinsic excitability. Crucial compartment for excitability regulation is the axon initial segment (AIS), as changes in AIS structure, but also voltage-gated ion channel type and density are known to occur during changes in activity and stringly influence action potential firing properties. However, the molecular mechanisms controlling AIS plasticity, and thereby allowing the AIS to dynamically regulate intrinsic excitability are still elusive.

Here, we show that the AIS scaffold AnkyrinG does not undergo strucural plasticity in cultured hippocampal neurons following 48 hours of network silencing. However, Patch-clamp recordings reveal that glutamatergic neurons exhibited a significant increase in their intrinsic excitability, contrary to GABAergic neurons. Using immunocytochemistry, we report that chronic silencing triggers an increase in Nav1.6 density at the AIS in excitatory neurons, and a decrease in inhibitory neurons. Furthermore, we find that that protein synthesis is needed for the upregulation of Nav1.6 channels in excitatory neurons, and observe an increase in SCN8A transcripts, encoding Nav1.6, specifically in these neurons. We also report a specific upregulation of Rbfox1, a transcription factor known to regulate SCN8A, in glutamatergic cells. Altogether, we reveal how Nav1.6 expression is regulated by changes in activity, allowing neurons to modulate their intrinsic excitability, thereby counterbalancing the alterations of network activity.

Dr Amélie Fréal

Amsterdam UMC, The Netherlands

Dr Amélie Fréal

Amsterdam UMC, The Netherlands

Dr Amélie Fréal is an Assistant Professor at the Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit Amsterdam and Amsterdam UMC, where she leads a research group studying the molecular mechanisms underlying neuronal excitability and plasticity. Her research focuses on the axon initial segment (AIS), the specialized neuronal domain where action potentials are initiated, and how this structure remodels in response to changes in neuronal activity.

Her lab combines human iPSC-derived neurons, primary neuronal cultures and brain slices with genome editing, advanced microscopy, proteomics, and electrophysiology to uncover how neurons maintain stable function while adapting to changing activity. She is particularly interested in how AIS plasticity contributes to neuronal homeostasis and how its disruption leads to neurological disease. Dr Fréal obtained her PhD in France and completed postdoctoral training in the Netherlands before establishing her independent research group. She is passionate about mentoring young scientists and fostering collaborative, interdisciplinary (and fun!) science.

Structural plasticity of the axon initial segment (AIS) strongly impacts the output of neurons and plays a fundamental role in the physiology and pathology of the brain. In the avian cochlear nucleus, where the AIS undergoes auditory experience-dependent shortening during development, we previously revealed that microtubule reorganisation via activation of CDK5 plays a key role in this process. However, the underlying molecular mechanisms are not fully understood.

Here we developed an in vivo CRISPR/Cas9 knockout platform using an all-in-one triple-guide RNA vector for delivery via electroporation and employed this approach to identify molecules involved in the developmental AIS shortening in the auditory circuits. We targeted fourteen molecules associated with microtubules and found that knockout of GSK3β impaired the AIS shortening, whereas overexpression of a constitutively active form of GSK3β facilitated the AIS shortening in vivo. In addition, this GSK3β-induced AIS shortening was reproduced in slice cultures and suppressed by microtubule stabilisation, supporting a role for GSK3β-dependent microtubule remodelling in the developmental AIS shortening. In this meeting, I will also present the effects of ankyrin-G knockout on spike output of neurons and discuss the role of the AIS in temporal coding in the brainstem auditory circuits.

Dr Hiroshi Kuba

Nagoya University, Japan

Dr Hiroshi Kuba

Nagoya University, Japan

Hiroshi Kuba is a professor of Cell Physiology at the Graduate School of Medicine, Nagoya University. He graduated from the Faculty of Medicine at Kyushu University in 1997 and completed two years of clinical training in neurosurgery. After getting his PhD in Harunori Ohmori’s lab at Kyoto University in 2003, he served as an academic staff in Kyoto University from 2003 to 2011. During this period, he also spent a year as a visiting fellow in Larry Trussell's lab in Oregon Health Science University (2009-2010) and got the present position in 2011. His research focuses on the mechanisms underlying the development and function of brainstem auditory circuits involved in sound localisation. His current interests include axon initial segment plasticity and neuro-glial interactions. He investigates these topics by combining multiple techniques, such as patch clamp recording, imaging, genome editing, and computer simulation.