Post-translational targeting of Rab35 by the effector IcsB of Shigella determines intracellular bacterial niche formation.

Escape from the bacterial-containing vacuole (BCV) is a key step of Shigella host cell invasion. Rab GTPases subverted to in situ-formed macropinosomes in the vicinity of the BCV have been shown to promote its rupture. The involvement of the BCV itself has remained unclear. We demonstrate that Rab35 is non-canonically entrapped at the BCV. Stimulated emission depletion imaging localizes Rab35 directly on the BCV membranes before vacuolar rupture. The bacterial effector IcsB, a lysine Nε-fatty acylase, is a key regulator of Rab35-BCV recruitment, and we show post-translational acylation of Rab35 by IcsB in its polybasic region. While Rab35 and IcsB are dispensable for the first step of BCV breakage, they are needed for the unwrapping of damaged BCV remnants from Shigella. This provides a framework for understanding Shigella invasion implicating re-localization of a Rab GTPase via its bacteria-dependent post-translational modification to support the mechanical unpeeling of the BCV.

Combating the Opioid Crisis and Its National Security Threat Through CReDO: A Multidisciplinary Solution With Disaster Medicine Implications.

For the first time in history, the United States surpassed 100 000 overdose-related deaths in a 12-month period, driven by synthetic opioids such as fentanyl. Also, for the first time, potential chemical weapons are readily available on the streets and the dark web. Opioids represent a rare trifecta, used for licit pain management, as an illicit drug of abuse, and with potential use as a weapon of terror. Community-based Response to Drug Overdose (CReDO) is an initiative to unite agencies, disciplines, government, and private partners in 1 coordinated opioid emergencies response plan under nationwide standards, and can be integrated into the disaster medicine discipline due to the risk of mass casualty incidents involving fentanyl or its derivatives. Attention to the opioid crisis through CReDO will save lives by promoting information sharing between disciplines, shortened response time to overdose clusters, community collaboration to identify criminal distribution networks, and holistic approaches to addiction.

Developmental emergence of two-stage nonlinear synaptic integration in cerebellar interneurons.

Synaptic transmission, connectivity, and dendritic morphology mature in parallel during brain development and are often disrupted in neurodevelopmental disorders. Yet how these changes influence the neuronal computations necessary for normal brain function are not well understood. To identify cellular mechanisms underlying the maturation of synaptic integration in interneurons, we combined patch-clamp recordings of excitatory inputs in mouse cerebellar stellate cells (SCs), three-dimensional reconstruction of SC morphology with excitatory synapse location, and biophysical modeling. We found that postnatal maturation of postsynaptic strength was homogeneously reduced along the somatodendritic axis, but dendritic integration was always sublinear. However, dendritic branching increased without changes in synapse density, leading to a substantial gain in distal inputs. Thus, changes in synapse distribution, rather than dendrite cable properties, are the dominant mechanism underlying the maturation of neuronal computation. These mechanisms favor the emergence of a spatially compartmentalized two-stage integration model promoting location-dependent integration within dendritic subunits.

A first-passage approach to diffusion-influenced reversible binding and its insights into nanoscale signaling at the presynapse.

Synaptic transmission between neurons is governed by a cascade of stochastic calcium ion reaction-diffusion events within nerve terminals leading to vesicular release of neurotransmitter. Since experimental measurements of such systems are challenging due to their nanometer and sub-millisecond scale, numerical simulations remain the principal tool for studying calcium-dependent neurotransmitter release driven by electrical impulses, despite the limitations of time-consuming calculations. In this paper, we develop an analytical solution to rapidly explore dynamical stochastic reaction-diffusion problems based on first-passage times. This is the first analytical model that accounts simultaneously for relevant statistical features of calcium ion diffusion, buffering, and its binding/unbinding reaction with a calcium sensor for synaptic vesicle fusion. In particular, unbinding kinetics are shown to have a major impact on submillisecond sensor occupancy probability and therefore cannot be neglected. Using Monte Carlo simulations we validated our analytical solution for instantaneous calcium influx and that through voltage-gated calcium channels. We present a fast and rigorous analytical tool that permits a systematic exploration of the influence of various biophysical parameters on molecular interactions within cells, and which can serve as a building block for more general cell signaling simulators.

Feed-forward recruitment of electrical synapses enhances synchronous spiking in the mouse cerebellar cortex.

In the cerebellar cortex, molecular layer interneurons use chemical and electrical synapses to form subnetworks that fine-tune the spiking output of the cerebellum. Although electrical synapses can entrain activity within neuronal assemblies, their role in feed-forward circuits is less well explored. By combining whole-cell patch-clamp and 2-photon laser scanning microscopy of basket cells (BCs), we found that classical excitatory postsynaptic currents (EPSCs) are followed by GABAA receptor-independent outward currents, reflecting the hyperpolarization component of spikelets (a synapse-evoked action potential passively propagating from electrically coupled neighbors). FF recruitment of the spikelet-mediated inhibition curtails the integration time window of concomitant excitatory postsynaptic potentials (EPSPs) and dampens their temporal integration. In contrast with GABAergic-mediated feed-forward inhibition, the depolarizing component of spikelets transiently increases the peak amplitude of EPSPs, and thus postsynaptic spiking probability. Therefore, spikelet transmission can propagate within the BC network to generate synchronous inhibition of Purkinje cells, which can entrain cerebellar output for driving temporally precise behaviors.

Distinct Nanoscale Calcium Channel and Synaptic Vesicle Topographies Contribute to the Diversity of Synaptic Function.

The nanoscale topographical arrangement of voltage-gated calcium channels (VGCC) and synaptic vesicles (SVs) determines synaptic strength and plasticity, but whether distinct spatial distributions underpin diversity of synaptic function is unknown. We performed single bouton Ca2+ imaging, Ca2+ chelator competition, immunogold electron microscopic (EM) localization of VGCCs and the active zone (AZ) protein Munc13-1, at two cerebellar synapses. Unexpectedly, we found that weak synapses exhibited 3-fold more VGCCs than strong synapses, while the coupling distance was 5-fold longer. Reaction-diffusion modeling could explain both functional and structural data with two strikingly different nanotopographical motifs: strong synapses are composed of SVs that are tightly coupled (∼10 nm) to VGCC clusters, whereas at weak synapses VGCCs were excluded from the vicinity (∼50 nm) of docked vesicles. The distinct VGCC-SV topographical motifs also confer differential sensitivity to neuromodulation. Thus, VGCC-SV arrangements are not canonical, and their diversity could underlie functional heterogeneity across CNS synapses.

Author Correction: Stability, affinity, and chromatic variants of the glutamate sensor iGluSnFR.

The version of this paper originally published cited a preprint version of ref. 12 instead of the published version (Proc. Natl. Acad. Sci. USA 115, 5594-5599; 2018), which was available before this Nature Methods paper went to press. The reference information has been updated in the PDF and HTML versions of the article.

Underpinning heterogeneity in synaptic transmission by presynaptic ensembles of distinct morphological modules.

Synaptic heterogeneity is widely observed but its underpinnings remain elusive. We addressed this issue using mature calyx of Held synapses whose numbers of bouton-like swellings on stalks of the nerve terminals inversely correlate with release probability (Pr). We examined presynaptic Ca2+ currents and transients, topology of fluorescently tagged knock-in Ca2+ channels, and Ca2+ channel-synaptic vesicle (SV) coupling distance using Ca2+ chelator and inhibitor of septin cytomatrix in morphologically diverse synapses. We found that larger clusters of Ca2+ channels with tighter coupling distance to SVs elevate Pr in stalks, while smaller clusters with looser coupling distance lower Pr in swellings. Septin is a molecular determinant of the differences in coupling distance. Supported by numerical simulations, we propose that varying the ensemble of two morphological modules containing distinct Ca2+ channel-SV topographies diversifies Pr in the terminal, thereby establishing a morpho-functional continuum that expands the coding capacity within a single synapse population.

Publisher Correction: Stability, affinity, and chromatic variants of the glutamate sensor iGluSnFR.

In the version of this paper originally published, important figure labels in Fig. 3d were not visible. An image layer present in the authors' original figure that included two small dashed outlines and text labels indicating ROI 1 and ROI 2, as well as a scale bar and the name of the cell label, was erroneously altered during image processing. The figure has been corrected in the HTML and PDF versions of the paper.

Stability, affinity, and chromatic variants of the glutamate sensor iGluSnFR.

Single-wavelength fluorescent reporters allow visualization of specific neurotransmitters with high spatial and temporal resolution. We report variants of intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) that are functionally brighter; detect submicromolar to millimolar amounts of glutamate; and have blue, cyan, green, or yellow emission profiles. These variants could be imaged in vivo in cases where original iGluSnFR was too dim, resolved glutamate transients in dendritic spines and axonal boutons, and allowed imaging at kilohertz rates.

Variations in Ca2+ Influx Can Alter Chelator-Based Estimates of Ca2+ Channel-Synaptic Vesicle Coupling Distance.

The timing and probability of synaptic vesicle fusion from presynaptic terminals is governed by the distance between voltage-gated Ca2+ channels (VGCCs) and Ca2+ sensors for exocytosis. This VGCC-sensor coupling distance can be determined from the fractional block of vesicular release by exogenous Ca2+ chelators, which depends on biophysical factors that have not been thoroughly explored. Using numerical simulations of Ca2+ reaction and diffusion, as well as vesicular release, we examined the contributions of conductance, density, and open duration of VGCCs, and the influence of endogenous Ca2+ buffers on the inhibition of exocytosis by EGTA. We found that estimates of coupling distance are critically influenced by the duration and amplitude of Ca2+ influx at active zones, but relatively insensitive to variations of mobile endogenous buffer. High concentrations of EGTA strongly inhibit vesicular release in close proximity (20-30 nm) to VGCCs if the flux duration is brief, but have little influence for longer flux durations that saturate the Ca2+ sensor. Therefore, the diversity in presynaptic action potential duration is sufficient to alter EGTA inhibition, resulting in errors potentially as large as 300% if Ca2+ entry durations are not considered when estimating VGCC-sensor coupling distances.SIGNIFICANT STATEMENT The coupling distance between voltage-gated Ca2+ channels and Ca2+ sensors for exocytosis critically determines the timing and probability of neurotransmitter release. Perfusion of presynaptic terminals with the exogenous Ca2+ chelator EGTA has been widely used for both qualitative and quantitative estimates of this distance. However, other presynaptic terminal parameters such as the amplitude and duration of Ca2+ entry can also influence EGTA inhibition of exocytosis, thus confounding conclusions based on EGTA alone. Here, we performed reaction-diffusion simulations of Ca2+-driven synaptic vesicle fusion, which delineate the critical parameters influencing an accurate prediction of coupling distance. Our study provides guidelines for characterizing and understanding how variability in coupling distance across chemical synapses could be estimated accurately.

Nicotine reverses hypofrontality in animal models of addiction and schizophrenia.

The prefrontal cortex (PFC) underlies higher cognitive processes that are modulated by nicotinic acetylcholine receptor (nAChR) activation by cholinergic inputs. PFC spontaneous default activity is altered in neuropsychiatric disorders, including schizophrenia-a disorder that can be accompanied by heavy smoking. Recently, genome-wide association studies (GWAS) identified single-nucleotide polymorphisms (SNPs) in the human CHRNA5 gene, encoding the α5 nAChR subunit, that increase the risks for both smoking and schizophrenia. Mice with altered nAChR gene function exhibit PFC-dependent behavioral deficits, but it is unknown how the corresponding human polymorphisms alter the cellular and circuit mechanisms underlying behavior. Here we show that mice expressing a human α5 SNP exhibit neurocognitive behavioral deficits in social interaction and sensorimotor gating tasks. Two-photon calcium imaging in awake mouse models showed that nicotine can differentially influence PFC pyramidal cell activity by nAChR modulation of layer II/III hierarchical inhibitory circuits. In α5-SNP-expressing and α5-knockout mice, lower activity of vasoactive intestinal polypeptide (VIP) interneurons resulted in an increased somatostatin (SOM) interneuron inhibitory drive over layer II/III pyramidal neurons. The decreased activity observed in α5-SNP-expressing mice resembles the hypofrontality observed in patients with psychiatric disorders, including schizophrenia and addiction. Chronic nicotine administration reversed this hypofrontality, suggesting that administration of nicotine may represent a therapeutic strategy for the treatment of schizophrenia, and a physiological basis for the tendency of patients with schizophrenia to self-medicate by smoking.

Differential Dendritic Integration of Synaptic Potentials and Calcium in Cerebellar Interneurons.

Dendritic voltage integration determines the transformation of synaptic inputs into output firing, while synaptic calcium integration drives plasticity mechanisms thought to underlie memory storage. Dendritic calcium integration has been shown to follow the same synaptic input-output relationship as dendritic voltage, but whether similar operations apply to neurons exhibiting sublinear voltage integration is unknown. We examined the properties and cellular mechanisms of these dendritic operations in cerebellar molecular layer interneurons using dendritic voltage and calcium imaging, in combination with synaptic stimulation or glutamate uncaging. We show that, while synaptic potentials summate sublinearly, concomitant dendritic calcium signals summate either linearly or supralinearly depending on the number of synapses activated. The supralinear dendritic calcium triggers a branch-specific, short-term suppression of neurotransmitter release that alters the pattern of synaptic activation. Thus, differential voltage and calcium integration permits dynamic regulation of neuronal input-output transformations without altering intrinsic nonlinear integration mechanisms.

A Role for Synaptic Input Distribution in a Dendritic Computation of Motion Direction in the Retina.

The starburst amacrine cell in the mouse retina presents an opportunity to examine the precise role of sensory input location on neuronal computations. Using visual receptive field mapping, glutamate uncaging, two-photon Ca(2+) imaging, and genetic labeling of putative synapses, we identify a unique arrangement of excitatory inputs and neurotransmitter release sites on starburst amacrine cell dendrites: the excitatory input distribution is skewed away from the release sites. By comparing computational simulations with Ca(2+) transients recorded near release sites, we show that this anatomical arrangement of inputs and outputs supports a dendritic mechanism for computing motion direction. Direction-selective Ca(2+) transients persist in the presence of a GABA-A receptor antagonist, though the directional tuning is reduced. These results indicate a synergistic interaction between dendritic and circuit mechanisms for generating direction selectivity in the starburst amacrine cell.

Optical control of NMDA receptors with a diffusible photoswitch.

N-methyl-D-aspartate receptors (NMDARs) play a central role in synaptic plasticity, learning and memory, and are implicated in various neuronal disorders. We synthesized a diffusible photochromic glutamate analogue, azobenzene-triazole-glutamate (ATG), which is specific for NMDARs and functions as a photoswitchable agonist. ATG is inactive in its dark-adapted trans-isoform, but can be converted into its active cis-isoform using one-photon (near UV) or two-photon (740 nm) excitation. Irradiation with violet light photo-inactivates ATG within milliseconds, allowing agonist removal on the timescale of NMDAR deactivation. ATG is compatible with Ca(2+) imaging and can be used to optically mimic synaptic coincidence detection protocols. Thus, ATG can be used like traditional caged glutamate compounds, but with the added advantages of NMDAR specificity, low antagonism of GABAR-mediated currents, and precise temporal control of agonist delivery.

Contribution of sublinear and supralinear dendritic integration to neuronal computations.

Nonlinear dendritic integration is thought to increase the computational ability of neurons. Most studies focus on how supralinear summation of excitatory synaptic responses arising from clustered inputs within single dendrites result in the enhancement of neuronal firing, enabling simple computations such as feature detection. Recent reports have shown that sublinear summation is also a prominent dendritic operation, extending the range of subthreshold input-output (sI/O) transformations conferred by dendrites. Like supralinear operations, sublinear dendritic operations also increase the repertoire of neuronal computations, but feature extraction requires different synaptic connectivity strategies for each of these operations. In this article we will review the experimental and theoretical findings describing the biophysical determinants of the three primary classes of dendritic operations: linear, sublinear, and supralinear. We then review a Boolean algebra-based analysis of simplified neuron models, which provides insight into how dendritic operations influence neuronal computations. We highlight how neuronal computations are critically dependent on the interplay of dendritic properties (morphology and voltage-gated channel expression), spiking threshold and distribution of synaptic inputs carrying particular sensory features. Finally, we describe how global (scattered) and local (clustered) integration strategies permit the implementation of similar classes of computations, one example being the object feature binding problem.

Synaptic diversity enables temporal coding of coincident multisensory inputs in single neurons.

The ability of the brain to rapidly process information from multiple pathways is critical for reliable execution of complex sensory-motor behaviors, yet the cellular mechanisms underlying a neuronal representation of multimodal stimuli are poorly understood. Here we explored the possibility that the physiological diversity of mossy fiber (MF) to granule cell (GC) synapses in the mouse vestibulocerebellum may contribute to the processing of coincident multisensory information at the level of individual GCs. We found that the strength and short-term dynamics of individual MF-GC synapses can act as biophysical signatures for primary vestibular, secondary vestibular and visual input pathways. Most GCs receive inputs from different modalities, which, when coactivated, produced enhanced GC firing rates and distinct first spike latencies. Thus, pathway-specific synaptic response properties permit temporal coding of correlated multisensory inputs by single GCs, thereby enriching sensory representation and facilitating pattern separation.

Ca2+ channel to synaptic vesicle distance accounts for the readily releasable pool kinetics at a functionally mature auditory synapse.

Precise regulation of synaptic vesicle (SV) release at the calyx of Held is critical for auditory processing. At the prehearing calyx of Held, synchronous and asynchronous release is mediated by fast and slow releasing SVs within the readily releasable pool (RRP). However, the posthearing calyx has dramatically different release properties. Whether developmental alterations in RRP properties contribute to the accelerated release time course found in posthearing calyces is not known. To study these questions, we performed paired patch-clamp recordings, deconvolution analysis, and numerical simulations of buffered Ca(2+) diffusion and SV release in postnatal day (P) 16-19 mouse calyces, as their release properties resemble mature calyces of Held. We found the P16-P19 calyx RRP consists of two pools: a fast pool (τ ≤ 0.9 ms) and slow pool (τ ∼4 ms), in which release kinetics and relative composition of the two pools were unaffected by 5 mm EGTA. Simulations of SV release from the RRP revealed that two populations of SVs were necessary to reproduce the experimental release rates: (1) SVs located close (∼5-25 nm) and (2) more distal (25-100 nm) to VGCC clusters. This positional coupling was confirmed by experiments showing 20 mm EGTA preferentially blocked distally coupled SVs. Lowering external [Ca(2+)] to in vivo levels reduced only the fraction SVs released from the fast pool. Therefore, we conclude that a dominant parameter regulating the mature calyx RRP release kinetics is the distance between SVs and VGCC clusters.

Nanoscale distribution of presynaptic Ca(2+) channels and its impact on vesicular release during development.

Synaptic efficacy and precision are influenced by the coupling of voltage-gated Ca(2+) channels (VGCCs) to vesicles. But because the topography of VGCCs and their proximity to vesicles is unknown, a quantitative understanding of the determinants of vesicular release at nanometer scale is lacking. To investigate this, we combined freeze-fracture replica immunogold labeling of Cav2.1 channels, local [Ca(2+)] imaging, and patch pipette perfusion of EGTA at the calyx of Held. Between postnatal day 7 and 21, VGCCs formed variable sized clusters and vesicular release became less sensitive to EGTA, whereas fixed Ca(2+) buffer properties remained constant. Experimentally constrained reaction-diffusion simulations suggest that Ca(2+) sensors for vesicular release are located at the perimeter of VGCC clusters (<30 nm) and predict that VGCC number per cluster determines vesicular release probability without altering release time course. This "perimeter release model" provides a unifying framework accounting for developmental changes in both synaptic efficacy and time course.