Drugs that inhibit TMEM16 proteins block SARS-CoV-2 spike-induced syncytia.

COVID-19 is a disease with unique characteristics that include lung thrombosis1, frequent diarrhoea2, abnormal activation of the inflammatory response3 and rapid deterioration of lung function consistent with alveolar oedema4. The pathological substrate for these findings remains unknown. Here we show that the lungs of patients with COVID-19 contain infected pneumocytes with abnormal morphology and frequent multinucleation. The generation of these syncytia results from activation of the SARS-CoV-2 spike protein at the cell plasma membrane level. On the basis of these observations, we performed two high-content microscopy-based screenings with more than 3,000 approved drugs to search for inhibitors of spike-driven syncytia. We converged on the identification of 83 drugs that inhibited spike-mediated cell fusion, several of which belonged to defined pharmacological classes. We focused our attention on effective drugs that also protected against virus replication and associated cytopathicity. One of the most effective molecules was the antihelminthic drug niclosamide, which markedly blunted calcium oscillations and membrane conductance in spike-expressing cells by suppressing the activity of TMEM16F (also known as anoctamin 6), a calcium-activated ion channel and scramblase that is responsible for exposure of phosphatidylserine on the cell surface. These findings suggest a potential mechanism for COVID-19 disease pathogenesis and support the repurposing of niclosamide for therapy.

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 obtained from male rat brains. Application of agonists to either AChRs generally, or mAChRs specifically, induced robust increases in network activity in the low-gamma range. Prolonged AChR stimulation for 48 h 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. Characterized by the presence of a strong M-current, the hyperadaptation phenotype was abolished by acute application of either M-channel antagonists or the reapplication 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.SIGNIFICANCE STATEMENT The large heterogeneity of neuron types in the brain, each with its own specific functional properties, provides the rich cellular tapestry needed to account for the vast diversity of behaviors. By studying the functional properties of neurons in the hippocampus, a region of the brain involved in learning and memory, we find that exposure to the neuromodulator acetylcholine can alter the relative number of functionally defined neuron types. Our findings suggest that the heterogeneity of neurons in the brain is not a static feature but can be modified by the ongoing activity of the circuits to which they belong.

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 characterized at the soma and proximal axon, knowledge of the propagation of APs toward distal axonal domains of mammalian CNS neurons remains limited. We used genetically encoded voltage indicators (GEVIs) to image APs with submillisecond temporal resolution simultaneously at different locations along the long axons of dissociated hippocampal neurons from rat embryos of either sex. We found that APs became sharper and showed remarkable fidelity as they traveled toward 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 serve to sustain AP fidelity, conveying a reliable digital signal to presynaptic boutons.SIGNIFICANCE STATEMENT The AP represents the electrical signal carried along axons toward distant presynaptic boutons where it culminates in the release of neurotransmitters. The nonlinearities involved in this process are such that small changes in AP shape can result in large changes in neurotransmitter release. Since axons are remarkably long structures, any distortions that APs suffer along the way have the potential to translate into a significant modulation of synaptic transmission, particularly in distal domains. To avoid these issues, distal axons have ensured that signals are kept remarkably constant and insensitive to modulation during a train, despite the long distances traveled. Here, we uncover the mechanisms that allow distal axonal domains to provide a reliable and faithful digital signal to presynaptic terminals.

Distance-dependent gradient in NMDAR-driven spine calcium signals along tapering dendrites.

Neurons receive a multitude of synaptic inputs along their dendritic arbor, but how this highly heterogeneous population of synaptic compartments is spatially organized remains unclear. By measuring N-methyl-d-aspartic acid receptor (NMDAR)-driven calcium responses in single spines, we provide a spatial map of synaptic calcium signals along dendritic arbors of hippocampal neurons and relate this to measures of synapse structure. We find that quantal NMDAR calcium signals increase in amplitude as they approach a thinning dendritic tip end. Based on a compartmental model of spine calcium dynamics, we propose that this biased distribution in calcium signals is governed by a gradual, distance-dependent decline in spine size, which we visualized using serial block-face scanning electron microscopy. Our data describe a cell-autonomous feature of principal neurons, where tapering dendrites show an inverse distribution of spine size and NMDAR-driven calcium signals along dendritic trees, with important implications for synaptic plasticity rules and spine function.

The role of spontaneous neurotransmission in synapse and circuit development.

In the past, the spontaneous release of neurotransmitter from presynaptic terminals has been thought of as a side effect of evoked release, with little functional significance. As our understanding of the process of spontaneous release has increased over time, this notion has gradually changed. In this review, we focus on the importance of this form of release during neuronal development, a time of extreme levels of plasticity that includes the growth of dendrites and axons as well as the formation of new synaptic contacts. This period also encompasses high levels of neurotransmitter release from growing axons, and recent studies have found that spontaneous transmitter release plays an important role in shaping neuronal morphology as well as modulating the properties of newly forming synaptic contacts in the brain. Here, we bring together the latest findings across different species to argue that the spontaneous release of neurotransmitter is an important player in the wiring of the brain during development.

Activity-dependent mismatch between axo-axonic synapses and the axon initial segment controls neuronal output.

The axon initial segment (AIS) is a structure at the start of the axon with a high density of sodium and potassium channels that defines the site of action potential generation. It has recently been shown that this structure is plastic and can change its position along the axon, as well as its length, in a homeostatic manner. Chronic activity-deprivation paradigms in a chick auditory nucleus lead to a lengthening of the AIS and an increase in neuronal excitability. On the other hand, a long-term increase in activity in dissociated rat hippocampal neurons results in an outward movement of the AIS and a decrease in the cell's excitability. Here, we investigated whether the AIS is capable of undergoing structural plasticity in rat hippocampal organotypic slices, which retain the diversity of neuronal cell types present at postnatal ages, including chandelier cells. These interneurons exclusively target the AIS of pyramidal neurons and form rows of presynaptic boutons along them. Stimulating individual CA1 pyramidal neurons that express channelrhodopsin-2 for 48 h leads to an outward shift of the AIS. Intriguingly, both the pre- and postsynaptic components of the axo-axonic synapses did not change position after AIS relocation. We used computational modeling to explore the functional consequences of this partial mismatch and found that it allows the GABAergic synapses to strongly oppose action potential generation, and thus downregulate pyramidal cell excitability. We propose that this spatial arrangement is the optimal configuration for a homeostatic response to long-term stimulation.

Activity-dependent relocation of the axon initial segment fine-tunes neuronal excitability.

In neurons, the axon initial segment (AIS) is a specialized region near the start of the axon that is the site of action potential initiation. The precise location of the AIS varies across and within different neuronal types, and has been linked to cells' information-processing capabilities; however, the factors determining AIS position in individual neurons remain unknown. Here we show that changes in electrical activity can alter the location of the AIS. In dissociated hippocampal cultures, chronic depolarization with high extracellular potassium moves multiple components of the AIS, including voltage-gated sodium channels, up to 17 mum away from the soma of excitatory neurons. This movement reverses when neurons are returned to non-depolarized conditions, and depends on the activation of T- and/or L-type voltage-gated calcium channels. The AIS also moved distally when we combined long-term LED (light-emitting diode) photostimulation with sparse neuronal expression of the light-activated cation channel channelrhodopsin-2; here, burst patterning of activity was successful where regular stimulation at the same frequency failed. Furthermore, changes in AIS position correlate with alterations in current thresholds for action potential spiking. Our results show that neurons can regulate the position of an entire subcellular structure according to their ongoing levels and patterns of electrical activity. This novel form of activity-dependent plasticity may fine-tune neuronal excitability during development.

Excitatory and inhibitory synapses show a tight subcellular correlation that weakens over development.

Neurons receive correlated levels of excitation and inhibition, a feature that is important for proper brain function. However, how this relationship between excitatory and inhibitory inputs is established during the dynamic period of circuit wiring 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 the dendrites of developing CA1 hippocampal neurons. This correlation was present within short dendritic stretches (<20 µm) 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 stabilizing 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.

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.

Independent vesicle pools underlie different modes of release during neuronal development.

Mature presynaptic terminals release neurotransmitter both in response to activity and spontaneously. We found that axons of rat hippocampal neurons initially show very high levels of exclusively spontaneous release, which progressively switches over to the mature phenotype during synapse formation. These two modes of vesicle cycling derive from distinct pools throughout development and the initiation of activity-dependent release was independent of postsynaptic contacts, suggesting it is an autonomous presynaptic event.

Chandelier cells shine a light on the formation of GABAergic synapses.

Uncovering the wiring rules employed by neurons during development represents a formidable challenge with important repercussions for neurodevelopmental disorders. Chandelier cells (ChCs) are a singular GABAergic interneuron type, with a unique morphology, that have recently begun to shed light on the rules that drive the formation and plasticity of inhibitory synapses. This review will focus on the wealth of recent data charting the emergence of synapses formed by ChCs onto pyramidal cells, from the molecules involved to the plasticity of these connections during development.

Aberrant axon initial segment plasticity and intrinsic excitability of ALS hiPSC motor neurons.

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 ALS human induced pluripotent stem cell (hiPSC) motor neurons. We find that early TDP-43 and C9orf72 hiPSC motor neurons show an increase in the length of the AIS and impaired activity-dependent AIS plasticity that is linked to abnormal homeostatic regulation of neuronal activity and intrinsic hyperexcitability. In turn, these hyperactive neurons drive increased spontaneous myofiber contractions of in vitro hiPSC motor units. In contrast, late hiPSC and postmortem ALS motor neurons show AIS shortening, and hiPSC motor neurons progress to hypoexcitability. At a molecular level, aberrant expression of the AIS master scaffolding protein ankyrin-G and AIS-specific voltage-gated sodium channels mirror these dynamic changes in AIS function and excitability. Our results point toward the AIS as an important site of dysfunction in ALS motor neurons.

Multi-synaptic boutons are a feature of CA1 hippocampal connections in the stratum oriens.

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 postnatal day 22 [P22] 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 with neighboring SSBs, features that were confirmed by super-resolution light microscopy. Computer simulations suggest that these properties favor synchronous activity in CA1 networks.

Corrigendum: Correlative Live-Cell and Super-Resolution Imaging to Link Presynaptic Molecular Organisation With Function.

[This corrects the article DOI: 10.3389/fnsyn.2022.830583.].

Correlative Live-Cell and Super-Resolution Imaging to Link Presynaptic Molecular Organisation With Function.

Information transfer at synapses occurs when vesicles fuse with the plasma membrane to release neurotransmitters, which then bind to receptors at the postsynaptic membrane. The process of neurotransmitter release varies dramatically between different synapses, but little is known about how this heterogeneity emerges. The development of super-resolution microscopy has revealed that synaptic proteins are precisely organised within and between the two parts of the synapse and that this precise spatiotemporal organisation fine-tunes neurotransmission. However, it remains unclear if variability in release probability could be attributed to the nanoscale organisation of one or several proteins of the release machinery. To begin to address this question, we have developed a pipeline for correlative functional and super-resolution microscopy, taking advantage of recent technological advancements enabling multicolour imaging. Here we demonstrate the combination of live imaging of SypHy-RGECO, a unique dual reporter that simultaneously measures presynaptic calcium influx and neurotransmitter release, with post hoc immunolabelling and multicolour single molecule localisation microscopy, to investigate the structure-function relationship at individual presynaptic boutons.

Synaptic activity and activity-dependent competition regulates axon arbor maturation, growth arrest, and territory in the retinotectal projection.

In the retinotectal projection, synapses guide retinal ganglion cell (RGC) axon arbor growth by promoting branch formation and by selectively stabilizing branches. To ask whether presynaptic function is required for this dual role of synapses, we have suppressed presynaptic function in single RGCs using targeted expression of tetanus toxin light-chain fused to enhanced green fluorescent protein (TeNT-Lc:EGFP). Time-lapse imaging of singly silenced axons as they arborize in the tectum of zebrafish larvae shows that presynaptic function is not required for stabilizing branches or for generating an arbor of appropriate complexity. However, synaptic activity does regulate two distinct aspects of arbor development. First, single silenced axons fail to arrest formation of highly dynamic but short-lived filopodia that are a feature of immature axons. Second, single silenced axons fail to arrest growth of established branches and so occupy significantly larger territories in the tectum than active axons. However, if activity-suppressed axons had neighbors that were also silent, axonal arbors appeared normal in size. A similar reversal in phenotype was observed when single TeNT-Lc:EGFP axons are grown in the presence of the NMDA receptor antagonist MK801 [(+)-5-methyl-10,11- dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate]. Although expansion of arbor territory is prevented when neighbors are silent, formation of transient filopodia is not. These results suggest that synaptic activity by itself regulates filopodia formation regardless of activity in neighboring cells but that the ability to arrest growth and focusing of axonal arbors in the target is an activity-dependent, competitive process.

Activity-dependent regulation of inhibitory synaptic transmission in hippocampal neurons.

Neural activity regulates the number and properties of GABAergic synapses in the brain, but the mechanisms underlying these changes are unclear. We found that blocking spike activity globally in developing hippocampal neurons from rats reduced the density of GABAergic terminals as well as the frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs). Chronic inactivity later in development led to a reduction in the mIPSC amplitude, without any change in GABAergic synapse density. By contrast, hyperpolarizing or abolishing spike activity in single neurons did not alter GABAergic synaptic inputs. Suppressing activity in individual presynaptic GABAergic neurons also failed to decrease synaptic output. Our results indicate that GABAergic synapses are regulated by the level of activity in surrounding neurons. Notably, we found that the expression of GABAergic plasticity involves changes in the amount of neurotransmitter in individual vesicles.

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

The 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.

Photocycles of channelrhodopsin-2.

Recent developments have used light-activated channels or transporters to modulate neuronal activity. One such genetically-encoded modulator of activity, channelrhodopsin-2 (ChR2), depolarizes neurons in response to blue light. In this work, we first conducted electrophysiological studies of the photokinetics of hippocampal cells expressing ChR2, for various light stimulations. These and other experimental results were then used for systematic investigation of the previously proposed three-state and four-state models of the ChR2 photocycle. We show the limitations of the previously suggested three-state models and identify a four-state model that accurately follows the ChR2 photocurrents. We find that ChR2 currents decay biexponentially, a fact that can be explained by the four-state model. The model is composed of two closed (C1 and C2) and two open (O1 and O2) states, and our simulation results suggest that they might represent the dark-adapted (C1-O1) and light-adapted (C2-O2) branches. The crucial insight provided by the analysis of the new model is that it reveals an adaptation mechanism of the ChR2 molecule. Hence very simple organisms expressing ChR2 can use this form of light adaptation.