In neurons, the axon initial segment (AIS) is a specialised region near the start of the axon that is the site of action potential initiation [1][2][3][4][5][6] . The precise location of the AIS varies across and within different neuronal types 7,8 , and has been linked to cells' information-processing capabilities 8 ; 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 potassium moves multiple components of the AIS, including voltage-gated sodium channels, up to 17 μm away from the soma of excitatory neurons. This movement reverses when neurons are returned to non-depolarized conditions, and depends upon 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.In dissociated hippocampal neurons, global depolarization with 15 mM extracellular potassium from 12 to 14 days in vitro (DIV) produced a significant distal shift in AIS location ( Fig. 1a-d and Supplementary Figs 1-3). Labelling for the AIS scaffolding protein ankyrin G 9 , showed that start, maximum, and end AIS positions were all significantly relocated away from the soma (Fig. 1a,b; start: Mann-Whitney U-test, P < 0.0001; maximum: P < 0.0001; end: P < 0.0001; n = 885 cells, 36 coverslips), leaving the length of the AIS unchanged ( Fig. 1a,b; P = 0.11). We also observed significant activity-dependent distal shifts in other AIS-specific proteins 10,11 , including the scaffolding protein βIV spectrin ( Fig. 1c,d; start: Mann-Whitney U-test, P < 0.0001; maximum: P < 0.0001; end: P < 0.0001; n = 1065 cells, 44 coverslips), the extracellular matrix binding protein neurofascin ( Fig. 1d; start: Mann-Whitney U-test, P < 0.0001; maximum: P = 0.002; end: P = 0.01; n = 96 cells, 4 coverslips), the ion channel-associated protein FGF14 ( Fig. 1d; start: MannWhitney U-test, P < 0.0001; maximum: P < 0.0001; end: P < 0.0001; n = 89 cells, 4 coverslips), and, vitally, the voltage-gated sodium channels (VGSCs) essential for actionCorrespondence and requests for materials should be addressed to M.G. (matthew.grubb@kcl.ac.uk) or J.B. (juan.burrone@kcl.ac.uk).. Author contributions M.G. planned and performed all experiments and analysis, and wrote the paper. J.B. produced simulation data, planned experiments,...

COVID-19 is a disease with unique characteristics that include lung thrombosis 1 , frequent diarrhoea 2 , abnormal activation of the inflammatory response 3 and rapid deterioration of lung function consistent with alveolar oedema 4 . 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.One of the defining features of coronavirus biology is the coordinated process by which the virus binds and enters the host cell, which involves both docking to receptors at the cell surface (ACE2 for SARS-CoV2 5 ), and proteolytic activation of the spike protein by host encoded proteases at two distinct sites 6 . One activation step is spike cleavage at the S1-S2 boundary, which can occur either before or after receptor binding. A second proteolytic activation exposes the S2 portion, and primes S2 for fusion of virus and cellular membranes. The protease priming event at this S2′ site and subsequent fusion can occur after endocytosis, in which cleavage is carried out by endosomal low pH-activated proteases such as cathepsin B and cathepsin L 7 , or at the plasma membrane, where cleavage can be mediated by TMPRSS2 [8][9][10] . The spike proteins of MERS-CoV and SARS-CoV-2 possess a multibasic amino acid sequence at the S1-S2 interface, which is not present in SARS-CoV 11 , that also allows cleavage by the ubiquitously expressed serine protease furin [12][13][14] . As a consequence, cells that express MERS-CoV and SARS-CoV-2 spike protein at the plasma membrane can fuse with other cells that express the respective receptors and form syncytia.

The rules by which neuronal activity causes long-term modification of synapses in the central nervous system are not fully understood. Whereas competitive or correlation-based rules result in local modification of synapses, homeostatic modifications allow neuron-wide changes in synaptic strength, promoting stability. Experimental investigations of these rules at central nervous system synapses have relied generally on manipulating activity in populations of neurons. Here, we investigated the effect of suppressing excitability in single neurons within a network of active hippocampal neurons by overexpressing an inward-rectifier potassium channel. Reducing activity in a neuron before synapse formation leads to a reduction in functional synaptic inputs to that neuron; no such reduction was observed when activity of all neurons was uniformly suppressed. In contrast, suppressing activity in a single neuron after synapses are established results in a homeostatic increase in synaptic input, which restores the activity of the neuron to control levels. Our results highlight the differences between global and selective suppression of activity, as well as those between early and late manipulation of activity.

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.

Synapses relay information through the release of neurotransmitters stored in presynaptic vesicles. The identity, kinetics and location of vesicle pools mobilized by neuronal activity have been studied using a variety of techniques. Here, we describe a novel genetically-encoded probe, biosyn, which consists of a biotinylated VAMP-2 expressed at presynaptic terminals. We exploit the high affinity interaction between streptavidin and biotin to label biosyn with fluorescent streptavidin during vesicle fusion. This approach allows tagging of vesicles sequentially, to visualize and establish the identity of presynaptic pools. Using this technique we were able to distinguish between two different pools of vesicles in rat hippocampal neurons: one that is released in response to presynaptic activity and another, distinct vesicle pool that spontaneously fuses with the plasma membrane. We further established that the spontaneous vesicles belong to a ‘resting pool’ that is normally not mobilized by neuronal activity and whose function is mostly unknown.