A longstanding goal in neuroscience has been to image membrane voltage across a population of individual neurons in an awake, behaving mammal. Here, we report a genetically encoded fluorescent voltage indicator, SomArchon, which exhibits millisecond response times and compatibility with optogenetic control, and which increases the sensitivity, signal-to-noise ratio, Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

BackgroundEnteric Escherichia coli survives the highly acidic environment of the stomach through multiple acid resistance (AR) mechanisms. The most effective system, AR2, decarboxylates externally-derived glutamate to remove cytoplasmic protons and excrete GABA. The first described system, AR1, does not require an external amino acid. Its mechanism has not been determined. The regulation of the multiple AR systems and their coordination with broader cellular metabolism has not been fully explored.ResultsWe utilized a combination of ChIP-Seq and gene expression analysis to experimentally map the regulatory interactions of four TFs: nac, ntrC, ompR, and csiR. Our data identified all previously in vivo confirmed direct interactions and revealed several others previously inferred from gene expression data. Our data demonstrate that nac and csiR directly modulate AR, and leads to a regulatory network model in which all four TFs participate in coordinating acid resistance, glutamate metabolism, and nitrogen metabolism. This model predicts a novel mechanism for AR1 by which the decarboxylation enzymes of AR2 are used with internally derived glutamate. This hypothesis makes several testable predictions that we confirmed experimentally.ConclusionsOur data suggest that the regulatory network underlying AR is complex and deeply interconnected with the regulation of GABA and glutamate metabolism, nitrogen metabolism. These connections underlie and experimentally validated model of AR1 in which the decarboxylation enzymes of AR2 are used with internally derived glutamate.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-016-0376-y) contains supplementary material, which is available to authorized users.

Highlights d One-photon calcium imaging of brain activity can suffer from neuropil crosstalk d Targeting GCaMPs to the cell body reduces neuropil crosstalk d One-photon imaging of somatic GCaMP reduces artifactual spikes and correlations d Somatic GCaMPs can be used in multiple species, such as mice and zebrafish

Fast, volumetric imaging over large scales has been a long-standing goal in biological microscopy. Scanning techniques such as fluorescence confocal microscopy can acquire 2D images at high resolution and high speed, but extending the acquisition to multiple planes at different depths requires an axial scanning mechanism that drastically reduces the acquisition speed. To address this challenge, we report an augmented variant of confocal microscopy where the key innovation consists to use a series of reflecting pinholes axially distributed in the detection plane, each one probing a different depth within the sample. As no axial scanning mechanism is involved, our technique provides simultaneous multiplane imaging over fields of view larger than a millimeter at video-rate. We demonstrate the general applicability of our technique to neuronal imaging of both Caenorhabditis elegans and mouse brains in-vivo.

Methods for one-photon fluorescent imaging of calcium dynamics in vivo are popular due to their ability to simultaneously capture the dynamics of hundreds of neurons across large fields of view, at a low equipment complexity and cost. In contrast to two-photon methods, however, one-photon methods suffer from higher levels of crosstalk between cell bodies and the surrounding neuropil, resulting in decreased signal-to-noise and artifactual correlations of neural activity. Here, we address this problem by engineering cell body-targeted variants of the fluorescent calcium indicator GCaMP6f. We screened fusions of GCaMP6f to both natural as well as engineered peptides, and identified fusions that localized GCaMP6f to within approximately 50 microns of the cell body of neurons in live mice and larval zebrafish. One-photon imaging of soma-targeted GCaMP6f in dense neural circuits reported fewer artifactual spikes from neuropil, increased signal-to-noise ratio, and decreased artifactual correlation across neurons. Thus, soma-targeting of fluorescent calcium indicators increases neuronal signal fidelity and may facilitate even greater usage of simple, powerful, one-photon methods of population imaging of neural calcium dynamics.

A longstanding goal in neuroscience has been to image membrane voltage, with high temporal precision and sensitivity, in awake behaving mammals. Here, we report a genetically encoded voltage indicator, SomArchon, which exhibits millisecond response times and compatibility with optogenetic control, and which increases the sensitivity, signal-to-noise ratio, and number of neurons observable, by manyfold over previous reagents. SomArchon only requires conventional one-photon microscopy to achieve these high performance characteristics. These improvements enable population analysis of neural activity, both at the subthreshold and spiking levels, in multiple brain regions -cortex, hippocampus, and striatum -of awake behaving mice. Using SomArchon, we detect both positive and negative responses of striatal neurons during movement, highlighting the power of voltage imaging to reveal bidirectional modulation. We also examine how the intracellular subthreshold theta oscillations of hippocampal neurons govern spike output, finding that nearby cells can exhibit highly correlated subthreshold activities, even as they generate highly divergent spiking patterns.

Summary Fibronectin intrabodies generated with mRNA display (FingRs) are a recently developed tool for labeling excitatory or inhibitory synapses, with the benefit of not altering endogenous synaptic protein expression levels or synaptic transmission. Here, we generated a viral vector FingR toolbox that allows for multi-color, neuron-type-specific labeling of excitatory or inhibitory synapses in multiple brain regions. We screened various fluorophores, FingR fusion configurations, and transcriptional control regulations in adeno-associated virus (AAV) and retrovirus vector designs. We report the development of a red FingR variant and demonstrated dual labeling of excitatory and inhibitory synapses in the same cells. Furthermore, we developed cre-inducible FingR AAV variants and demonstrated their utility, finding that the density of inhibitory synapses in aspiny striatal cholinergic interneurons remained unchanged in response to dopamine depletion. Finally, we generated FingR retroviral vectors, which enabled us to track the development of excitatory and inhibitory synapses in hippocampal adult-born granule cells.

Cross-modal interactions of auditory and visual temporal modulation were examined in a game-like experimental framework. Participants observed an audiovisual stimulus (an animated, sound-emitting fish) whose sound intensity and/or visual size oscillated sinusoidally at either 6 or 7 Hz. Participants made speeded judgments about the modulation rate in either the auditory or visual modality while doing their best to ignore information from the other modality. Modulation rate in the task-irrelevant modality matched the modulation rate in the task-relevant modality (congruent conditions), was at the other rate (incongruent conditions), or had no modulation (unmodulated conditions). Both performance accuracy and parameter estimates from drift-diffusion decision modeling indicated that (1) the presence of temporal modulation in both modalities, regardless of whether modulations were matched or mismatched in rate, resulted in audiovisual interactions; (2) congruence in audiovisual temporal modulation resulted in more reliable information processing; and (3) the effects of congruence appeared to be stronger when judging visual modulation rates (i.e., audition influencing vision), than when judging auditory modulation rates (i.e., vision influencing audition). The results demonstrate that audiovisual interactions from temporal modulations are bi-directional in nature, but with potential asymmetries in the size of the effect in each direction.