Single-cell Activity Research Articles

Functional architecture of reward learning in mushroom body extrinsic neurons of larval Drosophila

The brain adaptively integrates present sensory input, past experience, and options for future action. The insect mushroom body exemplifies how a central brain structure brings about such integration. Here we use a combination of systematic single-cell labeling, connectomics, transgenic silencing, and activation experiments to study the mushroom body at single-cell resolution, focusing on the behavioral architecture of its input and output neurons (MBINs and MBONs), and of the mushroom body intrinsic APL neuron. Our results reveal the identity and morphology of almost all of these 44 neurons in stage 3 Drosophila larvae. Upon an initial screen, functional analyses focusing on the mushroom body medial lobe uncover sparse and specific functions of its dopaminergic MBINs, its MBONs, and of the GABAergic APL neuron across three behavioral tasks, namely odor preference, taste preference, and associative learning between odor and taste. Our results thus provide a cellular-resolution study case of how brains organize behavior.

Neurons in the Endbrain of Numerically Naive Crows Spontaneously Encode Visual Numerosity

Endowed with an elaborate cerebral cortex, humans and other primates can assess the number of items ina set, or numerosity, from birth on [1] and without being trained [2]. Whether spontaneous numerosity extraction is a unique feat of the mammalian cerebral cortex [3-7] or rather an adaptive property that can be found in differently designed and independently evolved neural substrates, such as the avian enbrain[8], is unknown. To address this question, we recorded single-cell activity from the nidopallium caudolaterale (NCL), a high-level avian association brain area [9-11], of numerically naive crows. We found that a proportion of NCL neurons were spontaneously responsive to numerosity and tuned to the number of items, even though the crows were never trained to assess numerical quantity. Our data show that numerosity-selective neuronal responses are spontaneously present in the distinct endbrains of diverge vertebrate taxa. This seemingly hard-wired property of the avian endbrain to extract numerical quantity explains how birds in the wild, or right afterhatching, can exploit numerical cues when making foraging or social decisions. It suggests that endbrain circuitries that evolved based on convergent evolution, such as the avian endbrain, give rise to the same numerosity code.

Coding Schemes in the Archerfish Optic Tectum

Many studies have yielded valuable knowledge on the early visual system but it is biased since the studies have focused on terrestrial mammals alone. Here, to better account for visual systems in different environments and animal classes, we studied the structure of early visual processing in the archerfish which harnesses its extreme visual ability to hunt by shooting water jets at prey hanging on vegetation above the water. Thus, the archerfish provides a unique opportunity to study visual processing in a vertebrate which is an expert vision-guided predator with a very different brain structure than mammals. The receptive field structures in the archerfish (both sexes) optic tectum, the main visual processing region in the fish brain, were measured and linear non-linear cascades were used to analyze their properties. The findings indicate that the spatial receptive field structures lie on a continuum between circular and elliptical shapes. In addition, the cells' functional properties display a richness of response characteristics, since many cells could be captured by more than a single linear filter. Finally, the non-linear response functions that link linear filters and neuronal responses were found to be similar to the non-linear functions of models that describe terrestrial mammalian single cell activity. Overall our results help to better understand the early visual processing system across vertebrates.

Talking to cells: semiconductor nanomaterials at the cellular interface.

The interface of biological components with semiconductors is a growing field with numerous applications. For example, the interfaces can be used to sense and modulate the electrical activity of single cells and tissues. From the materials point of view, silicon is the ideal option for such studies due to its controlled chemical synthesis, scalable lithography for functional devices, excellent electronic and optical properties, biocompatibility and biodegradability. Recent advances in this area are pushing the bio-interfaces from the tissue and organ level to the single cell and sub-cellular regimes. In this progress report, we will describe some fundamental studies focusing on miniaturizing the bioelectric and biomechanical interfaces. Additionally, many of our highlighted examples involve freestanding silicon-based nanoscale systems, in addition to substrate-bound structures or devices; the former offers new promise for basic research and clinical application. In this report, we will describe recent developments in the interfacing of neuronal and cardiac cells and their networks. Moreover, we will briefly discuss the incorporation of semiconductor nanostructures for interfacing non-excitable cells in applications such as probing intracellular force dynamics and drug delivery. Finally, we will suggest several directions for future exploration.

Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs

Total RNA sequencing has been used to reveal poly(A) and non-poly(A) RNA expression, RNA processing and enhancer activity. To date, no method for full-length total RNA sequencing of single cells has been developed despite the potential of this technology for single-cell biology. Here we describe random displacement amplification sequencing (RamDA-seq), the first full-length total RNA-sequencing method for single cells. Compared with other methods, RamDA-seq shows high sensitivity to non-poly(A) RNA and near-complete full-length transcript coverage. Using RamDA-seq with differentiation time course samples of mouse embryonic stem cells, we reveal hundreds of dynamically regulated non-poly(A) transcripts, including histone transcripts and long noncoding RNA Neat1. Moreover, RamDA-seq profiles recursive splicing in >300-kb introns. RamDA-seq also detects enhancer RNAs and their cell type-specific activity in single cells. Taken together, we demonstrate that RamDA-seq could help investigate the dynamics of gene expression, RNA-processing events and transcriptional regulation in single cells.

Ketamine Alters Lateral Prefrontal Oscillations in a Rule-Based Working Memory Task.

Acute administration of N-methyl-D-aspartate receptor (NMDAR) antagonists in healthy humans and animals produces working memory deficits similar to those observed in schizophrenia. However, it is unclear whether they also lead to altered low-frequency (≤60 Hz) neural oscillatory activities similar to those associated with schizophrenia during working memory processes. Here, we recorded local field potentials (LFPs) and single-unit activity from the lateral prefrontal cortex (LPFC) of three male rhesus macaque monkeys while they performed a rule-based prosaccade and antisaccade working memory task both before and after systemic injections of a subanesthetic dose (≤0.7 mg/kg) of ketamine. Accompanying working-memory impairment, ketamine enhanced the low-gamma-band (30-60 Hz) and dampened the beta-band (13-30 Hz) oscillatory activities in the LPFC during both delay periods and intertrial intervals. It also increased task-related alpha-band activities, likely reflecting compromised attention. Beta-band oscillations may be especially relevant to working memory processes because stronger beta power weakly but significantly predicted shorter saccadic reaction time. Also in beta band, ketamine reduced the performance-related oscillation as well as the rule information encoded in the spectral power. Ketamine also reduced rule information in the spike field phase consistency in almost all frequencies up to 60 Hz. Our findings support NMDAR antagonists in nonhuman primates as a meaningful model for altered neural oscillations and synchrony, which reflect a disorganized network underlying the working memory deficits in schizophrenia.SIGNIFICANCE STATEMENT Low doses of ketamine, an NMDAR blocker, produce working memory deficits similar to those observed in schizophrenia. In the lateral prefrontal cortex, a key brain region for working memory, we found that ketamine altered neural oscillatory activities in similar ways that differentiate schizophrenic patients and healthy subjects during both task and nontask periods. Ketamine induced stronger gamma (30-60 Hz) and weaker beta (13-30 Hz) oscillations, reflecting local hyperactivity and reduced long-range communications. Furthermore, ketamine reduced performance-related oscillatory activities, as well as the rule information encoded in the oscillations and in the synchrony between single-cell activities and oscillations. The ketamine model helps link the molecular and cellular basis of neural oscillatory changes to the working memory deficit in schizophrenia.

Trigger versus Substrate: Multi-Scale Considerations for Arrhythmia Modulation by Pharmacological Action

Background: Cardiac arrhythmias such as ventricular fibrillation are intrinsically tissue-scale phenomena, wherein single-cell triggers (e.g. Ca2+-induced spontaneous depolarisation) must interact with tissue-level substrate (e.g. heterogeneous repolarisation) to result in complex and irregular organ excitation (e.g. self-perpetuating re-entrant excitation). Prolongation of the QT interval of the electrocardiogram (pQT), associated with conditions such as heart failure and long-QT syndromes, is linked to increased vulnerability to arrhythmia. Pharmacological management of arrhythmia associated with pQT has been demonstrated to have limited effectiveness; Understanding the impact of pharmacological modulation on the complex interaction between trigger and substrate remains a significant yet important challenge in order to improve intervention efficacy. Methods: We examined the efficacy of a hERG activator (MC-II-157c) to reduce the manifestation of cell- and tissue-scale triggers and its concomitant effects on the tissue substrate using a multi-scale modelling approach: a model of the human ventricular action potential was integrated with a model of stochastic 3D spatiotemporal Ca2+ dynamics, which was then coarse-grained for suitability for 1D-3D tissue simulations. Parameters were modified to mimic pQT and MC-II-157c conditions. Results: pQT conditions promoted the development of spontaneous release events underlying afterdepolarisations during rapid pacing. MC-II-157c applied to pQT conditions shortened the action potential duration, inhibited the development of afterdepolarisations and reduced the probability of afterdepolarisations manifesting as triggered activity in single cells and ectopic activity in tissue. However, it could also increase transmural dispersion of repolarisation, which manifested as an increased vulnerable window for unidirectional conduction block. Conclusions: The combination of stochastic release event modulation and transmural dispersion of repolarisation modulation by MC-II-157c resulted in an integrative behaviour wherein the arrhythmia trigger is reduced but the substrate is increased. Such variable overall vulnerability to arrhythmia cannot be predicted from single-cell studies alone.

Assessing protein activity in single cells

Assessing protein activity in single cells

Effect of a calcitonin gene-related peptide-binding L-RNA aptamer on neuronal activity in the rat spinal trigeminal nucleus

BackgroundCalcitonin gene-related peptide (CGRP) plays a major role in the pathogenesis of migraine and other primary headaches. Spinal trigeminal neurons integrate nociceptive afferent input from trigeminal tissues including intracranial afferents, and their activity is thought to reflect facial pain and headache in man. CGRP receptor inhibitors and anti-CGRP antibodies have been demonstrated to be therapeutically effective in migraine. In parallel, CGRP receptor inhibition has been shown to lower spinal trigeminal neuron activity in animal models of meningeal nociception.MethodsIn a rat model of meningeal nociception, single cell activity of neurons in the spinal trigeminal nucleus with meningeal afferent input was recorded to test a further pharmacological approach, scavenging CGRP with a CGRP-binding l-RNA oligonucleotide, the l-aptamer NOX-C89. Cumulative ascending doses of NOX-C89 were intravenously infused.ResultsSpontaneous activity of spinal trigeminal neurons did not change after 0.05 mg/kg NOX-C89, however, after additional infusion of 0.5 mg/kg and 5 mg/kg NOX-C89, spontaneous activity was dose-dependently reduced. Identical doses of a control l-aptamer had no effect. This pharmacological effect of NOX-C89 was observed 10–25 min after infusion, but no difference was detected in the period 0–5 min. For comparison, the previously investigated CGRP receptor antagonist olcegepant had reduced activity within 5 min after infusion. Alongside the reduced spontaneous activity, after infusion of NOX-C89 the heat-induced neuronal activity was abolished.ConclusionsScavenging CGRP by mirror-image RNA aptamers provides further evidence that this approach can be used to control spinal trigeminal activity.

Single Cell Heterogeneity is Absent in the Transgenic Paradigm of an Inherited Cataract

We have shown recently that highly variable transcriptional activity in single cortical fiber cells mediates the transition from nascent to terminally differentiated fiber cells in the developing ocular lens. The lens is composed of only two cell types, the anterior epithelium and the fiber cells derived from it; the fiber cell morphogenesis proceeds in an order that positions terminally differentiated oldest fiber cells in the center and the youngest nascent fiber cells, entering differentiation, at the periphery of the lens. This pattern of growth produces layers of cells, which when isolated as single cells retain their molecular and spatial context. Here, working with an inherited cataract paradigm, we demonstrate a remarkable absence of transcriptional heterogeneity concomitant with the absence of terminal differentiation. The spatial context of the data, obtained from single isolated fiber cells suggests that homogenization of gene activity may be a prelude to impending pathology when a mutation disrupts development. Funding: This work was supported the Gerald Oppenheimer Family Foundation for the Prevention of Eye Disease Endowment Fund and by the NIH grant (1R01EY024929) to SPB. Declaration of Interest: Authors declare no competing interests. Ethical Approval: All the work with animals followed the institutional guidelines and protocols approved by Animal Research Committee (UCLA).

Metabolic versatility of a novel N2 -fixing Alphaproteobacterium isolated from a marine oxygen minimum zone.

The N2 -fixing (diazotrophic) community in marine ecosystems is dominated by non-cyanobacterial microorganisms. Yet, very little is known about their identity, function and ecological relevance due to a lack of cultured representatives. Here we report a novel heterotrophic diazotroph isolated from the oxygen minimum zone (OMZ) off Peru. The new species belongs to the genus Sagittula (Rhodobacteraceae, Alphaproteobacteria) and its capability to fix N2 was confirmed in laboratory experiments. Genome sequencing revealed that it is a strict heterotroph with a high versatility in substrate utilization and energy acquisition mechanisms. Pathways for sulfide oxidation and nitrite reduction to nitrous oxide are encoded in the genome and might explain the presence throughout the Peruvian OMZ. The genome further indicates that this novel organism could be in direct interaction with other microbes or particles. NanoSIMS analyses were used to compare the metabolic potential of S. castanea with single-cell activity in situ; however, N2 fixation by this diazotroph could not be detected at the isolation site. While the biogeochemical impact of S. castanea is yet to be resolved, its abundance and widespread distribution suggests that its potential to contribute to the marine N input could be significant at a larger geographical scale.

Simultaneous electrical and contractile monitoring of cardiomyocytes on silicon microelectrode arrays

Simultaneous electrical and contractile monitoring of cardiomyocytes on silicon microelectrode arrays

Live-cell measurements of kinase activity in single cells using translocation reporters.

Although kinases are important regulators of many cellular processes, measuring their activity in live cells remains challenging. We have developed kinase translocation reporters (KTRs), which enable multiplexed measurements of the dynamics of kinase activity at a single-cell level. These KTRs are composed of an engineered construct in which a kinase substrate is fused to a bipartite nuclear localization signal (bNLS) and nuclear export signal (NES), as well as to a fluorescent protein for microscopy-based detection of its localization. The negative charge introduced by phosphorylation of the substrate is used to directly modulate nuclear import and export, thereby regulating the reporter's distribution between the cytoplasm and nucleus. The relative cytoplasmic versus nuclear fluorescence of the KTR construct (the C/N ratio) is used as a proxy for the kinase activity in living, single cells. Multiple KTRs can be studied in the same cell by fusing them to different fluorescent proteins. Here, we present a protocol to execute and analyze live-cell microscopy experiments using KTRs. We describe strategies for development of new KTRs and procedures for lentiviral expression of KTRs in a cell line of choice. Cells are then plated in a 96-well plate, from which multichannel fluorescent images are acquired with automated time-lapse microscopy. We provide detailed guidance for a computational analysis and parameterization pipeline. The entire procedure, from virus production to data analysis, can be completed in ∼10 d.

Motion contrast in primary visual cortex: a direct comparison of single neuron and population encoding.

Features from outside the classical receptive field (CRF) can modulate the stimulus-driven activity of single cells in the primary visual cortex. This modulation, mediated by horizontal and feedback networks, has been extensively described as a variation of firing rate and is considered the basis of processing features as, for example, motion contrast. However, surround influences have also been identified in pairwise spiking or local field coherence. Yet, evidence about co-existence and integration of different neural signatures remains elusive. To compare multiple signatures, we recorded spiking and LFP activity evoked by stimuli exhibiting a motion contrast in the CRFs surround in anesthetized cat primary visual cortex. We chose natural-like scenes over gratings to avoid predominance of simple visual features, which could be easily represented by a rate code. We analyzed firing rates and phase-locking to low-gamma frequency in single cells and neuronal assemblies. Motion contrast was reflected in all measures but in semi-independent populations. Whereas activation of assemblies accompanied single neuron rates, their phase relations were modulated differently. Interestingly, only assembly phase relations mirrored the direction of movement of the surround and were selectively affected by thermal deactivation of visual interhemispheric connections. We argue that motion contrast can be reflected in complementary and superimposed neuronal signatures that can represent different surround features in independent neuronal populations.

Axons of Individual Dorsal Horn Neurons Bifurcated to Project in Both the Anterolateral and the Postsynaptic Dorsal Column Systems

Sensory information stimulates receptors of somatosensory system neurons generating a signal that codifies the characteristics of peripheral stimulation. This information reaches the spinal cord and is relayed to supra-spinal structures through two main systems: the postsynaptic dorsal column-medial lemniscal (DC-ML) and the anterolateral (AL) systems. From the classical point of view, the DC-ML has an ipsilateral ascending pathway to the Gracilis (GRA) or Cuneate (CUN) nuclei and the AL has a contralateral ascending pathway to the ventral posterolateral (VPL) thalamic nucleus. These two systems have been the subject of multiple studies that established their independence and interactions. To analyze the ascending projections of L1–L5 spinal dorsal horn neurons in the rat, two retrograde neuronal tracers were injected into the GRA and the VPL. Additionally, an electrophysiological study was performed by applying electrical stimulation at the GRA or VPL and recording antidromic evoked activity in single unit spinal cord cells. Importantly, a subset of spinal dorsal horn neurons exhibited double staining, indicating that these neurons projected to both the GRA and the VPL. These double-stained neurons were located on both sides of the dorsal horn of the spinal cord. The spinal dorsal horn neurons exhibited antidromic and collision activities in response to both GRA and VPL electrical activation. These results show spinal cord neurons with bifurcated bilateral projections to both the DC-ML and AL systems. Based on these results, we named these neurons bilateral and bifurcated cells.

Combining Fluorescence and Bioluminescence Microscopy to Study the Series of Events from Cellular Signal Transduction to Gene Expression.

The molecular interactions and translocation of signal transduction factors in individual cells can be imaged by fluorescence microscopy. Alternatively, downstream promoter activity in single cells can be imaged by bioluminescence microscopy. However, the same stimuli can lead to different gene expression responses in individual cells. For this reason, it is desirable to simultaneously image signal transduction and gene expression events in the same cells. Here, we describe a method that combines fluorescence and bioluminescence microscopy to image protein kinase C (PKC) translocation from the cytosol to the plasma membrane and the expression of nuclear factor kappa-light polypeptide B (NF-κB)-regulated genes. © 2017 by John Wiley & Sons, Inc.

Caudal Intraparietal Sulcus and three-dimensional vision: A combined functional magnetic resonance imaging and single-cell study

The cortical network processing three-dimensional (3D) object structure defined by binocular disparity spans both the ventral and dorsal visual streams. However, very little is known about the neural representation of 3D structure at intermediate levels of the visual hierarchy. Here, we investigated the neural selectivity for 3D surfaces in the macaque Posterior Intraparietal area (PIP) in the medial bank of the caudal intraparietal sulcus (IPS). We first identified a region sensitive to depth-structure information in the medial bank of the caudal IPS using functional Magnetic Resonance Imaging (fMRI), and then recorded single-cell activity within this fMRI activation in the same animals. Most PIP neurons were selective for the 3D orientation of planar surfaces (first-order disparity) at very short latencies, whereas a very small fraction of PIP neurons were selective for curved surfaces (second-order disparity). A linear support vector machine classifier could reliably identify the direction of the disparity gradient in planar and curved surfaces based on the responses of a population of disparity-selective PIP neurons. These results provide the first detailed account of the neuronal properties in area PIP, which occupies an intermediate position in the hierarchy of visual areas involved in processing depth structure from disparity.

Trigger vs. Substrate: Multi-Dimensional Modulation of QT-Prolongation Associated Arrhythmic Dynamics by a hERG Channel Activator.

Background: Prolongation of the QT interval of the electrocardiogram (ECG), underlain by prolongation of the action potential duration (APD) at the cellular level, is linked to increased vulnerability to cardiac arrhythmia. Pharmacological management of arrhythmia associated with QT prolongation is typically achieved through attempting to restore APD to control ranges, reversing the enhanced vulnerability to Ca2+-dependent afterdepolarisations (arrhythmia triggers) and increased transmural dispersion of repolarisation (arrhythmia substrate) associated with APD prolongation. However, such pharmacological modulation has been demonstrated to have limited effectiveness. Understanding the integrative functional impact of pharmacological modulation requires simultaneous investigation of both the trigger and substrate.Methods: We implemented a multi-scale (cell and tissue) in silico approach using a model of the human ventricular action potential, integrated with a model of stochastic 3D spatiotemporal Ca2+ dynamics, and parameter modification to mimic prolonged QT conditions. We used these models to examine the efficacy of the hERG activator MC-II-157c in restoring APD to control ranges, examined its effects on arrhythmia triggers and substrates, and the interaction of these arrhythmia triggers and substrates.Results: QT prolongation conditions promoted the development of spontaneous release events underlying afterdepolarisations during rapid pacing. MC-II-157c applied to prolonged QT conditions shortened the APD, inhibited the development of afterdepolarisations and reduced the probability of afterdepolarisations manifesting as triggered activity in single cells. In tissue, QT prolongation resulted in an increased transmural dispersion of repolarisation, which manifested as an increased vulnerable window for uni-directional conduction block. In some cases, MC-II-157c further increased the vulnerable window through its effects on INa. The combination of stochastic release event modulation and transmural dispersion of repolarisation modulation by MC-II-157c resulted in an integrative behavior wherein the arrhythmia trigger is reduced but the arrhythmia substrate is increased, leading to variable and non-linear overall vulnerability to arrhythmia.Conclusion: The relative balance of reduced trigger and increased substrate underlies a multi-dimensional role of MC-II-157c in modulation of cardiac arrhythmia vulnerability associated with prolonged QT interval.

Dynamics of lipids and metabolites during the cell cycle of Chlamydomonas reinhardtii.

Metabolites and lipids are the final products of enzymatic processes, distinguishing the different cellular functions and activities of single cells or whole tissues. Understanding these cellular functions within a well-established model system requires a systemic collection of molecular and physiological information. In the current report, the green alga Chlamydomonas reinhardtii was selected to establish a comprehensive workflow for the detailed multi-omics analysis of a synchronously growing cell culture system. After implementation and benchmarking of the synchronous cell culture, a two-phase extraction method was adopted for the analysis of proteins, lipids, metabolites and starch from a single sample aliquot of as little as 10-15million Chlamydomonas cells. In a proof of concept study, primary metabolites and lipids were sampled throughout the diurnal cell cycle. The results of these time-resolved measurements showed that single compounds were not only coordinated with each other in different pathways, but that these complex metabolic signatures have the potential to be used as biomarkers of various cellular processes. Taken together, the developed workflow, including the synchronized growth of the photoautotrophic cell culture, in combination with comprehensive extraction methods and detailed metabolic phenotyping has the potential for use in in-depth analysis of complex cellular processes, providing essential information for the understanding of complex biological systems.

Monitoring Photosynthetic Activity in Microalgal Cells by Raman Spectroscopy with Deuterium Oxide as a Tracking Probe.

Microalgae offer great potential for the production of biofuel, but high photosynthetic activity is demanded for the practical realisation of microalgal biofuels. To this end, it is essential to evaluate the photosynthetic activity of single microalgal cells in a heterogeneous population. In this study, we present a method to monitor the photosynthetic activity of microalgae (in particular Euglena gracilis, a microalgal species of unicellular, photosynthetic, flagellate protists as our model organism) at single-cell resolution by Raman spectroscopy with deuterium from deuterium oxide (D2 O) as a tracking probe. Specifically, we replaced H2 O in culture media with D2 O up to a concentration of 20 % without disturbing the growth rate of E. gracilis cells and evaluated C-D bond formation as a consequence of photosynthetic reactions by Raman spectroscopy. We used the probe to monitor the kinetics of the C-D bond formation in E. gracilis cells by incubating them in D2 O media under light irradiation. Furthermore, we demonstrated Raman microscopy imaging of each single E. gracilis cell to discriminate deuterated cells from normal cells. Our results hold great promise for Raman-based screening of E. gracilis and potentially other microalgae with high photosynthetic activity by using D2 O as a tracking probe.