Biology subjects

Burrone, J.

Publications and source records attributed to Burrone, J..

14 recordsLinked to original sources

bioRxiv2025

U7 small nuclear RNA splice-switching therapeutics for STMN2 and UNC13A in Amyotrophic Lateral Sclerosis

TDP-43 nuclear depletion in amyotrophic lateral sclerosis (ALS) causes de-repression of cryptic exons (CEs) in multiple transcripts, including UNC13A and STMN2, disrupting synaptic transmission and neurite outgrowth. We developed a therapeutic U7 snRNA (tU7) approach that suppresses TDP-43-dependent mis-splicing, restores target gene expression, rescues neuronal functions in human iPSC-derived neurons, and shows target engagement in vivo, positioning tU7-mediated splicing correction as a promising therapeutic strategy for ALS.

neuroscience↗

bioRxiv2025

Increased HCN1 activity in human excitatory neurons drives excessive network bursting in Dravet syndrome

Dravet syndrome (DS) is a severe childhood epilepsy caused by mutations of the sodium channel NaV1.1. These mutations are thought to compromise the ability of inhibitory interneurons to regulate network activity, leading to seizure events. However, standard treatments to restore inhibition have limited efficacy, suggesting the existence of additional pathological mechanisms. Here, we use hiPSC-derived neuronal networks containing both excitatory and inhibitory neurons to show that excessive bursting activity in DS cultures is driven by excitatory neurons. This rise in bursting frequency is caused by the increased expression of HCN1 "pacemaker" channels in excitatory neurons, and bursting activity can be normalised using a channel blocker. With this work, we propose a new pathophysiological mechanism in DS and identify HCN1 as a novel therapeutic target.

neuroscience↗

bioRxiv2025

Unreliable homeostatic action potential broadening in cultured dissociated neurons

Homeostatic plasticity preserves neuronal activity against perturbations. Recently, somatic action potential broadening was proposed as a key homeostatic adaptation to chronic inactivity in neocortical neurons. Since action potential shape critically controls calcium entry and neuronal function, broadening provides an attractive homeostatic feedback mechanism to regulate activity. Here, we report that chronic inactivity induced by sodium channel block does not broaden action potentials in neocortical neurons under a wide range of conditions. In contrast, action potentials were broadened in CA3 neurons of organotypic hippocampal cultures by chronic sodium channel block and in hippocampal dissociated cultures by chronic synaptic block. Mechanistically, BK-type potassium channels were proposed to underly inactivity-induced action potential broadening. However, BK channels did not affect action potential duration in our recordings. Our results indicate that action potential broadening can occur in specific neurons and conditions but is not a general mechanism of homeostatic plasticity in cultured neurons.

neuroscience↗

bioRxiv2024

In vivo modulation of network activity drives the nanoscale reorganisation of axo-axonic synapses at the axon initial segment

Chemical synapses control their strength through the nanoscale clustering of postsynaptic receptors into sub-synaptic domains (SSDs). Despite their importance in synapse function, the properties and plasticity of these domains are not well understood in vivo, particularly in inhibitory synapses. We used direct Stochastic Optical Resolution Microscopy (dSTORM) to show that Gephyrin, the main inhibitory receptor scaffold protein, is organised into SSDs in vivo, with distinct arrangements depending on their sub-cellular location and presynaptic partner. Furthermore, chronic chemogenetic increases in cortical activity caused a reduction in Gephyrin SSD volume specifically in axo-axonic, but not axo-dendritic, synapses. Functionally, this resulted in a weakening of axo-axonic contacts. We show that the nanoscale arrangement of synapses in the brain is plastic and used to fine-tune synaptic gain in vivo.

neuroscience↗

bioRxiv2024

Semi-automated navigation for efficient targeting of electron tomography to regions of interest in volume correlative light and electron microscopy

Electron microscopy is essential for the quantitative study of synaptic ultrastructure. At present, the correlation of functional and structural properties of the same synapse is extremely challenging. We introduce a novel integrated workflow designed to simplify sample navigation across spatial scales, allowing the identification of individual synapses from optical microscopy mouse brain image stacks that can be targeted for analysis using electron tomography imaging. We developed a software which has a function to register multimodal images using a novel segmentation-based image registration algorithm as well as a function to visualize all the registration results. Using our newly designed software we streamline mapping of high-resolution optical imaging onto reference maps using blood vessels as endogenous fiducial marks. Further we demonstrate significant improvements on the ultramicrotomy stage of volume Correlative Light and Electron Microscopy (vCLEM) workflows, providing real time guidance to targeted trimming to match previously acquired Regions Of Interest (ROIs), and reliable estimates of cutting depth relative to ROI, based on fluorescence imaging of TEM ready ultrathin sections. Using this workflow, we successfully targeted TEM tomography to the proximal axonal region containing the Axon Initial Segment identified using fluorescent light microscopy.

neuroscience↗

bioRxiv2024

Loss of TDP-43 induces synaptic dysfunction that is rescued by UNC13A splice-switching ASOs

TDP-43 loss of function induces multiple splicing changes, including a cryptic exon in the amyotrophic lateral sclerosis and fronto-temporal lobar degeneration risk gene UNC13A, leading to nonsense-mediated decay of UNC13A transcripts and loss of protein. UNC13A is an active zone protein with an integral role in coordinating pre-synaptic function. Here, we show TDP-43 depletion induces a severe reduction in synaptic transmission, leading to an asynchronous pattern of network activity. We demonstrate that these deficits are largely driven by a single cryptic exon in UNC13A. Antisense oligonucleotides targeting the UNC13A cryptic exon robustly rescue UNC13A protein levels and restore normal synaptic function, providing a potential new therapeutic approach for ALS and other TDP-43-related disorders.

neuroscience↗

bioRxiv2024

Excitatory and inhibitory synapses form a tight subcellular balance along dendrites that decorrelates over development

A balance between excitation and inhibition is crucial for neurotypical brain function. Indeed, disruptions in this relationship are frequently associated with the pathophysiology of neurodevelopmental disorders. Nevertheless, how this balance is established during the dynamic period of neurodevelopment remains unexplored. Using multiple techniques, including in utero electroporation, electron microscopy and electrophysiology, we reveal a tight correlation in the distribution of excitatory and inhibitory synapses along dendrites of developing CA1 hippocampal neurons. This balance was present within short dendritic stretches (<20{micro}m), and surprisingly, was most pronounced during early development, sharply declining with maturity. The tight matching between excitation and inhibition was unexpected, as inhibitory synapses lacked an active zone when formed and exhibited compromised evoked release. We propose that inhibitory synapses form as a stabilising scaffold, to counterbalance growing excitation levels. This relationship diminishes over time, suggesting a critical role for a subcellular balance in early neuronal function and circuit formation.

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bioRxiv2023

Spatial subdomains in the Optic Tectum for the encoding of visual information

1Neurons across the visual system provide estimates of the visual features they encode. However, the reliability of those estimates can vary across the neuronal population. Here, we use information theory to provide a spatial map of how well neurons can distinguish ethologically-relevant visual stimuli across the entire larval zebrafish optic tectum, a brain region responsible for driving visually guided behaviour. We find that the ability of neurons to discriminate between stimuli is non-uniformly distributed across the tectum. Specifically, we show that information about local motion is preferentially encoded in the posterior tectum, whilst information about whole-field motion is preferentially encoded in the anterior tectum. This is achieved through two systematic changes along the anterior-posterior axis of the tectum: (i) a change in the number of neurons that discriminate between stimuli and (ii) a change in how well each neuron can discriminate between stimuli. By classifying neurons into distinct subtypes based on their response properties we uncovered a small group of neurons that are spatially localised to specific regions of the tectum and are able to discriminate between visual stimuli in a highly reliable manner. Our results highlight the importance of implementing information theoretic approaches to assess visual responses and provide a novel description of regional specialisation in the zebrafish optic tectum.

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bioRxiv2022

Multi-synaptic boutons are a feature of CA1 hippocampal connections that may underlie network synchrony

Excitatory synapses are typically described as single synaptic boutons (SSBs), where one presynaptic bouton contacts a single postsynaptic spine. Using serial section block face scanning electron microscopy, we found that this textbook definition of the synapse does not fully apply to the CA1 region of the hippocampus. Roughly half of all excitatory synapses in the stratum oriens involved multi-synaptic boutons (MSBs), where a single presynaptic bouton containing multiple active zones contacted many postsynaptic spines (from 2 to 7) on the basal dendrites of different cells. The fraction of MSBs increased during development (from P21 to P100) and decreased with distance from the soma. Curiously, synaptic properties such as active zone (AZ) or postsynaptic density (PSD) size exhibited less within-MSB variation when compared to neighbouring SSBs, features that were confirmed by super-resolution light microscopy. Computer simulations suggest that these properties favour synchronous activity in CA1 networks.

neuroscience↗

bioRxiv2022

Pathogenic TDP-43 Disrupts Axon Initial Segment Structure and Neuronal Excitability in a Human iPSC Model of ALS

Dysregulated neuronal excitability is a hallmark of amyotrophic lateral sclerosis (ALS). We sought to investigate how functional changes to the axon initial segment (AIS), the site of action potential generation, could impact neuronal excitability in a human iPSC model of ALS. We found that early (6-week) ALS-related TDP-43G298S motor neurons showed an increase in the length of the AIS, relative to CRISPR-corrected controls. This was linked to neuronal hyperexcitability and increased spontaneous contractions of hiPSC-myofibers in compartmentalised neuromuscular co-cultures. In contrast late (10-week) TDP-43G298S motor neurons showed reduced AIS length and hypoexcitability. At a molecular level aberrant expression of the AIS master scaffolding protein Ankyrin-G, and the AIS-specific voltage-gated ion channels SCN1A (Nav1.1) and SCN8A (Nav1.6) mirrored these dynamic changes in excitability. Finally, at all stages, TDP-43G298S motor neurons showed compromised activity-dependent plasticity of the AIS, further contributing to abnormal excitability. Our results point toward the AIS as an important subcellular target driving changes to neuronal excitability in ALS.

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bioRxiv2022

Cholinergic stimulation modulates the functional composition of CA3 cell types in the hippocampus

The functional heterogeneity of hippocampal CA3 pyramidal neurons has emerged as a key aspect of circuit function. Here, we explored the effects of long-term cholinergic activity on the functional heterogeneity of CA3 pyramidal neurons in organotypic slices. Application of agonists to either acetylcholine receptors (AChRs) generally, or muscarinic AChRs (mAChRs) specifically, induced robust increases in network activity in the low-gamma range. Prolonged AChR stimulation for 48 hrs uncovered a population of hyperadapting CA3 pyramidal neurons that typically fired a single, early action potential in response to current injection. Although these neurons were present in control networks, their proportions were dramatically increased following long-term cholinergic activity. Characterised by the presence of a strong M-current, the hyperadaptation phenotype was abolished by acute application of either M-channel antagonists or the re-application of AChR agonists. We conclude that long-term mAChR activation modulates the intrinsic excitability of a subset of CA3 pyramidal cells, uncovering a highly plastic cohort of neurons that are sensitive to chronic ACh modulation. Our findings provide evidence for the activity-dependent plasticity of functional heterogeneity in the hippocampus.

neuroscience↗

bioRxiv2020

Voltage-gated potassium channels ensure action potential shape fidelity in distal axons

The initiation and propagation of the action potential (AP) along an axon allows neurons to convey information rapidly and across distant sites. Although AP properties have typically been characterised at the soma and proximal axon, the propagation of APs towards distal axonal domains of mammalian neurons remains limited. We used Genetically Encoded Voltage Indicators (GEVIs) to image APs simultaneously at different locations along the long axons of dissociated hippocampal neurons with sub-millisecond temporal resolution. We found that APs became sharper and showed remarkable fidelity as they traveled towards distal axons, even during a high frequency train. Blocking voltage-gated potassium channels (Kv) with 4-AP resulted in an increase in AP width in all compartments, which was stronger at distal locations and exacerbated during AP trains. We conclude that the higher levels of Kv channel activity in distal axons serves to sustain AP fidelity, conveying a reliable digital signal to presynaptic boutons.

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bioRxiv2018

Homeostatic plasticity rules control the wiring of axo-axonic synapses at the axon initial segment

GABAergic interneurons are chiefly responsible for controlling the activity of local circuits in the cortex1,2. However, the rules that govern the wiring of interneurons are not well understood3. Chandelier cells (ChCs) are a type of GABAergic interneuron that control the output of hundreds of neighbouring pyramidal cells through axo-axonic synapses which target the axon initial segment (AIS)4. Despite their importance in modulating circuit activity, our knowledge of the development and function of axo-axonic synapses remains elusive. In this study, we investigated the role of activity in the formation and plasticity of ChC synapses. 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 resulted in a reversible decrease in axo-axonic connections. Voltage imaging of GABAergic transmission at the AIS showed that axo-axonic synapses were depolarising during this period. Identical manipulations of network activity in older mice (P40-P46), when ChC synapses are inhibitory, resulted in an increase in axo-axonic synapses. We propose that the direction of ChC plasticity follows homeostatic rules that depend on the polarity of axo-axonic synapses.

neuroscience↗

bioRxiv2017

Activity-Dependent Control Of Inhibitory Interneuron Number In The Mammalian Cortex

Cortical networks are composed of excitatory projection neurons and inhibitory interneurons. Finding the right balance between the two is important for controlling overall cortical excitation and network dynamics. However, it is unclear how the correct number of cortical interneurons (CIs) is established in the mammalian forebrain. CIs are generated in excess from basal forebrain progenitors and their final numbers are adjusted via an intrinsically determined program of apoptosis that takes place during an early postnatal window. Here, we provide evidence that the extent of CI apoptosis during this critical period is plastic, cell type specific and can be reduced in a cell autonomous manner by acute increases in neuronal activity. We propose that the physiological state of the emerging neural network controls the activity levels of local CIs to modulate their numbers in a homeostatic manner.

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