Two facts about the hippocampus have been common currency among neuroscientists for several decades. First, lesions of the hippocampus in humans prevent the acquisition of new episodic memories; second, activity-dependent synaptic plasticity is a prominent feature of hippocampal synapses. Given this background, the hypothesis that hippocampus-dependent memory is mediated, at least in part, by hippocampal synaptic plasticity has seemed as cogent in theory as it has been difficult to prove in practice. Here we argue that the recent development of transgenic molecular devices will encourage a shift from mechanistic investigations of synaptic plasticity in single neurons towards an analysis of how networks of neurons encode and represent memory, and we suggest ways in which this might be achieved. In the process, the hypothesis that synaptic plasticity is necessary and sufficient for information storage in the brain may finally be validated.

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.

Dscam is an immunoglobulin (Ig) superfamily member that regulates axon guidance and targeting in Drosophila. Alternative splicing potentially generates 38,016 isoforms differing in their extracellular Ig and transmembrane domains. We demonstrate that Dscam mediates the sorting of axons in the developing mushroom body (MB). This correlates with the precise spatiotemporal pattern of Dscam protein expression. We demonstrate that MB neurons express different arrays of Dscam isoforms and that single MB neurons express multiple isoforms. Two different Dscam isoforms differing in their extracellular domains introduced as transgenes into single mutant cells partially rescued the mutant phenotype. Expression of one isoform of Dscam in a cohort of MB neurons induced dominant phenotypes, while expression of a single isoform in a single cell did not. We propose that different extracellular domains of Dscam share a common function and that differences in isoforms expressed on the surface of neighboring axons influence interactions between them.

1. The kinetics of exocytosis and endocytosis were studied in the giant synaptic terminal of depolarizing bipolar cells from the goldfish retina. Two techniques were applied: capacitance measurements of changes in membrane surface area, and fluorescence measurements of exocytosis using the membrane dye FM1_43. 2. Three phases of exocytosis occurred during maintained depolarization to 0 mV. The first component was complete within about 10 ms and involved a pool of 1200-1800 vesicles (with a total membrane area equivalent to about 1·6% of the surface of the terminal). The second component of exocytosis involved the release of about 4400 vesicles over 1 s. The third component of exocytosis was stimulated continuously at a rate of about 1000 vesicles s¢. 3. After short depolarizations (< 200 ms), neither the FM1_43 signal nor the capacitance signal continued to rise, indicating that exocytosis stopped rapidly after closure of Ca¥ channels. The fall in capacitance could therefore be used to monitor endocytosis independently of exocytosis. The capacitance measured after brief stimuli began to fall immediately, recovering to the pre-stimulus baseline with a rate constant of 0·8 s¢. 4. The amount of exocytosis measured using the capacitance and FM1_43 techniques was similar during the first 200 ms of depolarization, suggesting that the most rapidly released vesicles could be detected by either method. 5. After a few seconds of continuous stimulation, the net increase in membrane surface area reached a plateau at about 5%, even though continuous exocytosis occurred at a rate of 0·9 % s¢. Under these conditions of balanced exocytosis and endocytosis, the rate constant of endocytosis was about 0·2 s¢. The average rate of endocytosis during maintained depolarization was therefore considerably slower than the rate observed after a brief stimulus. 6. After longer depolarizations (> 500 ms), both the capacitance and FM1_43 signals continued to rise for periods of seconds after closure of Ca¥ channels. The continuation of exocytosis was correlated with a persistent increase in [Ca¥]é in the synaptic terminal, as indicated by the activation of a Ca¥-dependent conductance and measurements of [Ca¥]é using the fluorescent indicator furaptra. 7. The delayed fall in membrane capacitance after longer depolarizations occurred along a double exponential time course indicating the existence of two endocytic processes: fast endocytosis, with a rate constant of 0·8 s¢, and slow endocytosis, with a rate constant of 0·1 s¢. 8. Increasing the duration of depolarization caused an increase in the fraction of membrane recovered by slow endocytosis. After a 100 ms stimulus, all the membrane was recycled by fast endocytosis, but after a 5 s depolarization, about 50 % of the membrane was recycled by slow endocytosis. 9. These results demonstrate the existence of fast and slow endocytic mechanisms at a synapse and support the idea that prolonged stimulation leads to an increase in the amount of membrane retrieved by the slower route. The rise in cyt...

The Drosophila melanogaster gene Dscam is essential for axon guidance and has 38,016 possible alternative splice forms. This diversity can potentially be used to distinguish cells. We analyzed the Dscam mRNA isoforms expressed by different cell types and individual cells. The choice of splice variants expressed is regulated both spatially and temporally. Different subtypes of photoreceptors express broad yet distinctive spectra of Dscam isoforms. Single-cell RT-PCR documented that individual cells express several different Dscam isoforms and allowed an estimation of the diversity that is present. For example, we estimate that each R3/R4 photoreceptor cell expresses 14-50 distinct mRNAs chosen from the spectrum of thousands of splice variants distinctive of its cell type. Thus, the Dscam repertoire of each cell is different from those of its neighbors, providing a potential mechanism for generating unique cell identity in the nervous system and elsewhere. To circumvent these technical difficulties, we used an approach that combined a customized oligonucleotide microarray, isolation of distinct populations of cells and sensitive RT-PCR that allowed examination of Dscam expression by single cells. Here we show that Dscam alternative splicing is differentially regulated both during development and in different tissues. A given cell type expresses a broad, yet distinctive, spectrum of splice variants. Individual cells express a unique repertoire of Dscam splice variants. We estimate that individual R3/R4 photoreceptors express 14-50 distinct mRNA molecules chosen from a subset of thousands of Dscam isoforms characteristic of the population. This would ensure that the Dscam repertoire of each cell is different from those of its neighbors. A R T I C L E S RESULTS A microarray for analyzing Dscam alternative splicingThe genomic structure of Dscam 1 is shown in Figure 1a. There are four alternatively spliced exons. In this study we focused on three alternatively spliced exons, which may create many possible isoforms (Fig. 1a): the cluster in exon 4 has 12 alternative exons, the cluster in exon 6 has 48 alternative exons and the cluster in exon 9 has 33 alternative exons. Each of these highly variable exons can form a portion of an immunoglobulin domain in the extracellular region of the protein. We designed a microarray containing probes for all 93 alternative exons in these three clusters. We amplified cloned DNAs (whose identities were confirmed by sequencing) by PCR to create a reference library that equally represents all the alternative exons present in the microarray. To test the specificity of the different probes in the microarray, we used combinations of RNAs in controlled amounts (Fig. 1b). RNA samples were in vitro transcribed from cloned cDNAs containing different combinations of exons 4, 6 and 9. We carried out a quantitative analysis of four independent experiments in which eight different samples were combined in different amounts spanning two orders of magnitude. Throughout this study, we expressed sample...

Calcium-triggered exocytosis at the synapse is suppressed by addition of calcium chelators, but the effects of endogenous Ca(2+) buffers have not been tested. We find that 80% of Ca(2+) binding sites in the synaptic terminal of retinal bipolar cells were associated with mobile molecules that suppressed activation of Ca(2+)-sensitive K(+) channels with an efficiency equivalent to approximately 1.2 mM BAPTA. Removing these buffers caused a 30-fold increase in the number of vesicles released by Ca(2+) tail currents lasting approximately 0.5 ms and a 2-fold increase in the rapidly releasable pool of vesicles (RRP). The effects of BAPTA and EGTA indicate that vesicles comprising the RRP were docked at variable distances from Ca(2+) channels. We propose that endogenous Ca(2+) buffers regulate the size of the RRP by suppressing the release of vesicles toward the periphery of the active zone.

To investigate the regulation of endocytosis by Ca 2؉ , we have made capacitance measurements in the synaptic terminal of depolarizing bipolar cells from the retina of goldfish. After a brief depolarization, all of the excess membrane was retrieved rapidly ( Ϸ1 s). But when the rise in free [Ca 2؉ ] was reduced by the introduction of Ca 2؉ buffers [1,2-bis(2-aminophenoxy)ethane-N,N,N ,N -tetraacetate (BAPTA) or EGTA], a large fraction of the membrane was retrieved by a second, slower mechanism ( > 10 s). The block of fast endocytosis by EGTA could be overcome by increasing the amplitude of the Ca 2؉ current, demonstrating that Ca 2؉ influx was the trigger for fast endocytosis. These manipulations of the Ca 2؉ signal altered the relative proportions of fast and slow endocytosis but did not modulate the rate constants of these processes. A brief stimulus that triggered fast endocytosis did not generate a significant rise in the spatially averaged [Ca 2؉ ], indicating that Ca 2؉ regulated endocytosis through an action close to the active zone. The slow mode of retrieval occurred at the resting [Ca 2؉ ]. These results demonstrate that Ca 2؉ influx couples fast endocytosis and exocytosis at this synapse.

Highlights d Axo-axonic synapses and Chandelier cell axons develop rapidly between P12 and P18 d Increasing network activity during this period reduces axoaxonic synapse number d The depolarizing nature of axo-axonic synapses suggests this plasticity is homeostatic d When axo-axonic synapses become hyperpolarizing, synapse plasticity is reversed