Ensemble dynamics and information flow deduction from whole-brain imaging data.

The recent advancements in large-scale activity imaging of neuronal ensembles offer valuable opportunities to comprehend the process involved in generating brain activity patterns and understanding how information is transmitted between neurons or neuronal ensembles. However, existing methodologies for extracting the underlying properties that generate overall dynamics are still limited. In this study, we applied previously unexplored methodologies to analyze time-lapse 3D imaging (4D imaging) data of head neurons of the nematode Caenorhabditis elegans. By combining time-delay embedding with the independent component analysis, we successfully decomposed whole-brain activities into a small number of component dynamics. Through the integration of results from multiple samples, we extracted common dynamics from neuronal activities that exhibit apparent divergence across different animals. Notably, while several components show common cooperativity across samples, some component pairs exhibited distinct relationships between individual samples. We further developed time series prediction models of synaptic communications. By combining dimension reduction using the general framework, gradient kernel dimension reduction, and probabilistic modeling, the overall relationships of neural activities were incorporated. By this approach, the stochastic but coordinated dynamics were reproduced in the simulated whole-brain neural network. We found that noise in the nervous system is crucial for generating realistic whole-brain dynamics. Furthermore, by evaluating synaptic interaction properties in the models, strong interactions within the core neural circuit, variable sensory transmission and importance of gap junctions were inferred. Virtual optogenetics can be also performed using the model. These analyses provide a solid foundation for understanding information flow in real neural networks.

WormTensor: a clustering method for time-series whole-brain activity data from C. elegans.

BACKGROUND: In the field of neuroscience, neural modules and circuits that control biological functions have been found throughout entire neural networks. Correlations in neural activity can be used to identify such neural modules. Recent technological advances enable us to measure whole-brain neural activity with single-cell resolution in several species including [Formula: see text]. Because current neural activity data in C. elegans contain many missing data points, it is necessary to merge results from as many animals as possible to obtain more reliable functional modules. RESULTS: In this work, we developed a new time-series clustering method, WormTensor, to identify functional modules using whole-brain activity data from C. elegans. WormTensor uses a distance measure, modified shape-based distance to account for the lags and the mutual inhibition of cell-cell interactions and applies the tensor decomposition algorithm multi-view clustering based on matrix integration using the higher orthogonal iteration of tensors (HOOI) algorithm (MC-MI-HOOI), which can estimate both the weight to account for the reliability of data from each animal and the clusters that are common across animals. CONCLUSION: We applied the method to 24 individual C. elegans and successfully found some known functional modules. Compared with a widely used consensus clustering method to aggregate multiple clustering results, WormTensor showed higher silhouette coefficients. Our simulation also showed that WormTensor is robust to contamination from noisy data. WormTensor is freely available as an R/CRAN package https://cran.r-project.org/web/packages/WormTensor .

Stiffness of primordial germ cells is required for their extravasation in avian embryos.

Unlike mammals, primordial germ cells (PGCs) in avian early embryos exploit blood circulation to translocate to the somatic gonadal primordium, but how circulating PGCs undergo extravasation remains elusive. We demonstrate with single-cell level live-imaging analyses that the PGCs are arrested at a specific site in the capillary plexus, which is predominantly governed by occlusion at a narrow path in the vasculature. The occlusion is enabled by a heightened stiffness of the PGCs mediated by actin polymerization. Following the occlusion, PGCs reset their stiffness to soften in order to squeeze through the endothelial lining as they transmigrate. Our discovery also provides a model for the understanding of metastasizing cancer extravasation occurring mainly by occlusion.

3DeeCellTracker, a deep learning-based pipeline for segmenting and tracking cells in 3D time lapse images.

Despite recent improvements in microscope technologies, segmenting and tracking cells in three-dimensional time-lapse images (3D + T images) to extract their dynamic positions and activities remains a considerable bottleneck in the field. We developed a deep learning-based software pipeline, 3DeeCellTracker, by integrating multiple existing and new techniques including deep learning for tracking. With only one volume of training data, one initial correction, and a few parameter changes, 3DeeCellTracker successfully segmented and tracked ~100 cells in both semi-immobilized and 'straightened' freely moving worm's brain, in a naturally beating zebrafish heart, and ~1000 cells in a 3D cultured tumor spheroid. While these datasets were imaged with highly divergent optical systems, our method tracked 90-100% of the cells in most cases, which is comparable or superior to previous results. These results suggest that 3DeeCellTracker could pave the way for revealing dynamic cell activities in image datasets that have been difficult to analyze.

Microscopes have been used to decrypt the tiny details of life since the 17th century. Now, the advent of 3D microscopy allows scientists to build up detailed pictures of living cells and tissues. In that effort, automation is becoming increasingly important so that scientists can analyze the resulting images and understand how bodies grow, heal and respond to changes such as drug therapies. In particular, algorithms can help to spot cells in the picture (called cell segmentation), and then to follow these cells over time across multiple images (known as cell tracking). However, performing these analyses on 3D images over a given period has been quite challenging. In addition, the algorithms that have already been created are often not user-friendly, and they can only be applied to a specific dataset gathered through a particular scientific method. As a response, Wen et al. developed a new program called 3DeeCellTracker, which runs on a desktop computer and uses a type of artificial intelligence known as deep learning to produce consistent results. Crucially, 3DeeCellTracker can be used to analyze various types of images taken using different types of cutting-edge microscope systems. And indeed, the algorithm was then harnessed to track the activity of nerve cells in moving microscopic worms, of beating heart cells in a young small fish, and of cancer cells grown in the lab. This versatile tool can now be used across biology, medical research and drug development to help monitor cell activities.

Neuron ID dataset facilitates neuronal annotation for whole-brain activity imaging of C. elegans.

BACKGROUND: Annotation of cell identity is an essential process in neuroscience that allows comparison of cells, including that of neural activities across different animals. In Caenorhabditis elegans, although unique identities have been assigned to all neurons, the number of annotatable neurons in an intact animal has been limited due to the lack of quantitative information on the location and identity of neurons. RESULTS: Here, we present a dataset that facilitates the annotation of neuronal identities, and demonstrate its application in a comprehensive analysis of whole-brain imaging. We systematically identified neurons in the head region of 311 adult worms using 35 cell-specific promoters and created a dataset of the expression patterns and the positions of the neurons. We found large positional variations that illustrated the difficulty of the annotation task. We investigated multiple combinations of cell-specific promoters driving distinct fluorescence and generated optimal strains for the annotation of most head neurons in an animal. We also developed an automatic annotation method with human interaction functionality that facilitates annotations needed for whole-brain imaging. CONCLUSION: Our neuron ID dataset and optimal fluorescent strains enable the annotation of most neurons in the head region of adult C. elegans, both in full-automated fashion and a semi-automated version that includes human interaction functionalities. Our method can potentially be applied to model species used in research other than C. elegans, where the number of available cell-type-specific promoters and their variety will be an important consideration.

SPF-CellTracker: Tracking Multiple Cells with Strongly-Correlated Moves Using a Spatial Particle Filter.

Tracking many cells in time-lapse 3D image sequences is an important challenging task of bioimage informatics. Motivated by a study of brain-wide 4D imaging of neural activity in C. elegans, we present a new method of multi-cell tracking. Data types to which the method is applicable are characterized as follows: (i) cells are imaged as globular-like objects, (ii) it is difficult to distinguish cells on the basis of shape and size only, (iii) the number of imaged cells in the several-hundred range, (iv) movements of nearly-located cells are strongly correlated, and (v) cells do not divide. We developed a tracking software suite that we call SPF-CellTracker. Incorporating dependency on the cells' movements into the prediction model is the key for reducing the tracking errors: the cell switching and the coalescence of the tracked positions. We model the target cells' correlated movements as a Markov random field and we also derive a fast computation algorithm, which we call spatial particle filter. With the live-imaging data of the nuclei of C. elegans neurons in which approximately 120 nuclei of neurons were imaged, the proposed method demonstrated improved accuracy compared to the standard particle filter and the method developed by Tokunaga et al. (2014).

Environmental responsiveness of tubulin glutamylation in sensory cilia is regulated by the p38 MAPK pathway.

Glutamylation is a post-translational modification found on tubulin that can alter the interaction between microtubules (MTs) and associated proteins. The molecular mechanisms regulating tubulin glutamylation in response to the environment are not well understood. Here, we show that in the sensory cilia of Caenorhabditis elegans, tubulin glutamylation is upregulated in response to various signals such as temperature, osmolality, and dietary conditions. Similarly, tubulin glutamylation is modified in mammalian photoreceptor cells following light adaptation. A tubulin glutamate ligase gene ttll-4, which is essential for tubulin glutamylation of axonemal MTs in sensory cilia, is activated by p38 MAPK. Amino acid substitution of TTLL-4 has revealed that a Thr residue (a putative MAPK-phosphorylation site) is required for enhancement of tubulin glutamylation. Intraflagellar transport (IFT), a bidirectional trafficking system specifically observed along axonemal MTs, is required for the formation, maintenance, and function of sensory cilia. Measurement of the velocity of IFT particles revealed that starvation accelerates IFT, which was also dependent on the Thr residue of TTLL-4. Similarly, starvation-induced attenuation of avoidance behaviour from high osmolality conditions was also dependent on ttll-4. Our data suggest that a novel evolutionarily conserved regulatory system exists for tubulin glutamylation in sensory cilia in response to the environment.

An improved inverse-type Ca2+ indicator can detect putative neuronal inhibition in Caenorhabditis elegans by increasing signal intensity upon Ca2+ decrease.

Sensory processing is regulated by the coordinated excitation and inhibition of neurons in neuronal circuits. The analysis of neuronal activities has greatly benefited from the recent development of genetically encoded Ca2+ indicators (GECIs). These molecules change their fluorescence intensities or colours in response to changing levels of Ca2+ and can, therefore, be used to sensitively monitor intracellular Ca2+ concentration, which enables the detection of neuronal excitation, including action potentials. These GECIs were developed to monitor increases in Ca2+ concentration; therefore, neuronal inhibition cannot be sensitively detected by these GECIs. To overcome this difficulty, we hypothesised that an inverse-type of GECI, whose fluorescence intensity increases as Ca2+ levels decrease, could sensitively monitor reducing intracellular Ca2+ concentrations. We, therefore, developed a Ca2+ indicator named inverse-pericam 2.0 (IP2.0) whose fluorescent intensity decreases 25-fold upon Ca2+ binding in vitro. Using IP2.0, we successfully detected putative neuronal inhibition by monitoring the decrease in intracellular Ca2+ concentration in AWCON and ASEL neurons in Caenorhabditis elegans. Therefore, IP2.0 is a useful tool for studying neuronal inhibition and for the detailed analysis of neuronal activities in vivo.

Gonadal Maturation Changes Chemotaxis Behavior and Neural Processing in the Olfactory Circuit of Caenorhabditis elegans.

Many animal species change their behavior according to their stage of development. However, the mechanisms involved in translating their developmental stage into the modifications of the neuronal circuits that underlie these behavioral changes remain unknown. Here we show that Caenorhabditis elegans changes its olfactory preferences during development. Larvae exhibit a weak chemotactic response to the food-associated odor diacetyl, whereas adults exhibit a strong response. We show that germline loss, caused either by laser ablation of germline precursor cells or mutations, results in a diacetyl-specific chemotactic defect in adult animals. These results suggest that germline cells, which proliferate dramatically during the larval stages, enhance chemotaxis to diacetyl. Removal experiments of specific neurons suggested that AWA olfactory neurons and their downstream interneurons, AIA and AIB, are required for germline-dependent chemotactic enhancement. Calcium imaging in animals lacking germline cells indicates that the neural responses of AWA and AIB to diacetyl stimuli are decreased compared with animals with an intact germline. These changes in neural activities may at least partly explain the behavioral change of animals lacking germline cells. Furthermore, this germline-dependent chemotactic change depends on the transcription factor DAF-16/FOXO. We find that organismal behavior changes throughout development by integrating information about physiological status from internal tissues to modify a simple sensory circuit.

Accurate Automatic Detection of Densely Distributed Cell Nuclei in 3D Space.

To measure the activity of neurons using whole-brain activity imaging, precise detection of each neuron or its nucleus is required. In the head region of the nematode C. elegans, the neuronal cell bodies are distributed densely in three-dimensional (3D) space. However, no existing computational methods of image analysis can separate them with sufficient accuracy. Here we propose a highly accurate segmentation method based on the curvatures of the iso-intensity surfaces. To obtain accurate positions of nuclei, we also developed a new procedure for least squares fitting with a Gaussian mixture model. Combining these methods enables accurate detection of densely distributed cell nuclei in a 3D space. The proposed method was implemented as a graphical user interface program that allows visualization and correction of the results of automatic detection. Additionally, the proposed method was applied to time-lapse 3D calcium imaging data, and most of the nuclei in the images were successfully tracked and measured.

Automated detection and tracking of many cells by using 4D live-cell imaging data.

MOTIVATION: Automated fluorescence microscopes produce massive amounts of images observing cells, often in four dimensions of space and time. This study addresses two tasks of time-lapse imaging analyses; detection and tracking of the many imaged cells, and it is especially intended for 4D live-cell imaging of neuronal nuclei of Caenorhabditis elegans. The cells of interest appear as slightly deformed ellipsoidal forms. They are densely distributed, and move rapidly in a series of 3D images. Thus, existing tracking methods often fail because more than one tracker will follow the same target or a tracker transits from one to other of different targets during rapid moves. RESULTS: The present method begins by performing the kernel density estimation in order to convert each 3D image into a smooth, continuous function. The cell bodies in the image are assumed to lie in the regions near the multiple local maxima of the density function. The tasks of detecting and tracking the cells are then addressed with two hill-climbing algorithms. The positions of the trackers are initialized by applying the cell-detection method to an image in the first frame. The tracking method keeps attacking them to near the local maxima in each subsequent image. To prevent the tracker from following multiple cells, we use a Markov random field (MRF) to model the spatial and temporal covariation of the cells and to maximize the image forces and the MRF-induced constraint on the trackers. The tracking procedure is demonstrated with dynamic 3D images that each contain >100 neurons of C.elegans. AVAILABILITY: http://daweb.ism.ac.jp/yoshidalab/crest/ismb2014 SUPPLEMENTARY INFORMATION: Supplementary data are available at http://daweb.ism.ac.jp/yoshidalab/crest/ismb2014

Forgetting in C. elegans is accelerated by neuronal communication via the TIR-1/JNK-1 pathway.

The control of memory retention is important for proper responses to constantly changing environments, but the regulatory mechanisms underlying forgetting have not been fully elucidated. Our genetic analyses in C. elegans revealed that mutants of the TIR-1/JNK-1 pathway exhibited prolonged retention of olfactory adaptation and salt chemotaxis learning. In olfactory adaptation, conditioning induces attenuation of odor-evoked Ca(2+) responses in olfactory neurons, and this attenuation is prolonged in the TIR-1/JNK-1-pathway mutant animals. We also found that a pair of neurons in which the pathway functions is required for the acceleration of forgetting, but not for sensation or adaptation, in wild-type animals. In addition, the neurosecretion from these cells is important for the acceleration of forgetting. Therefore, we propose that these neurons accelerate forgetting through the TIR-1/JNK-1 pathway by sending signals that directly or indirectly stimulate forgetting.

An expanded palette of genetically encoded Ca²âº indicators.

Engineered fluorescent protein (FP) chimeras that modulate their fluorescence in response to changes in calcium ion (Ca(2+)) concentration are powerful tools for visualizing intracellular signaling activity. However, despite a decade of availability, the palette of single FP-based Ca(2+) indicators has remained limited to a single green hue. We have expanded this palette by developing blue, improved green, and red intensiometric indicators, as well as an emission ratiometric indicator with an 11,000% ratio change. This series enables improved single-color Ca(2+) imaging in neurons and transgenic Caenorhabditis elegans. In HeLa cells, Ca(2+) was imaged in three subcellular compartments, and, in conjunction with a cyan FP-yellow FP-based indicator, Ca(2+) and adenosine 5'-triphosphate were simultaneously imaged. This palette of indicators paints the way to a colorful new era of Ca(2+) imaging.

A novel zf-MYND protein, CHB-3, mediates guanylyl cyclase localization to sensory cilia and controls body size of Caenorhabditis elegans.

Cilia are important sensory organelles, which are thought to be essential regulators of numerous signaling pathways. In Caenorhabditis elegans, defects in sensory cilium formation result in a small-body phenotype, suggesting the role of sensory cilia in body size determination. Previous analyses suggest that lack of normal cilia causes the small-body phenotype through the activation of a signaling pathway which consists of the EGL-4 cGMP-dependent protein kinase and the GCY-12 receptor-type guanylyl cyclase. By genetic suppressor screening of the small-body phenotype of a cilium defective mutant, we identified a chb-3 gene. Genetic analyses placed chb-3 in the same pathway as egl-4 and gcy-12 and upstream of egl-4. chb-3 encodes a novel protein, with a zf-MYND motif and ankyrin repeats, that is highly conserved from worm to human. In chb-3 mutants, GCY-12 guanylyl cyclase visualized by tagged GFP (GCY-12::GFP) fails to localize to sensory cilia properly and accumulates in cell bodies. Our analyses suggest that decreased GCY-12 levels in the cilia of chb-3 mutants may cause the suppression of the small-body phenotype of a cilium defective mutant. By observing the transport of GCY-12::GFP particles along the dendrites to the cilia in sensory neurons, we found that the velocities and the frequencies of the particle movement are decreased in chb-3 mutant animals. How membrane proteins are trafficked to cilia has been the focus of extensive studies in vertebrates and invertebrates, although only a few of the relevant proteins have been identified. Our study defines a new regulator, CHB-3, in the trafficking process and also shows the importance of ciliary targeting of the signaling molecule, GCY-12, in sensory-dependent body size regulation in C. elegans. Given that CHB-3 is highly conserved in mammal, a similar system may be used in the trafficking of signaling proteins to the cilia of other species.

Magnesium excretion in C. elegans requires the activity of the GTL-2 TRPM channel.

Systemic magnesium homeostasis in mammals is primarily governed by the activities of the TRPM6 and TRPM7 cation channels, which mediate both uptake by the intestinal epithelial cells and reabsorption by the distal convoluted tubule cells in the kidney. In the nematode, C. elegans, intestinal magnesium uptake is dependent on the activities of the TRPM channel proteins, GON-2 and GTL-1. In this paper we provide evidence that another member of the TRPM protein family, GTL-2, acts within the C. elegans excretory cell to mediate the excretion of excess magnesium. Thus, the activity of GTL-2 balances the activities of the paralogous TRPM channel proteins, GON-2 and GTL-1.

A calcium wave mediated by gap junctions coordinates a rhythmic behavior in C. elegans.

Intercellular calcium waves can be observed in adult tissues, but whether they are instructive, permissive, or even required for behavior is predominantly unknown. In the nematode Caenorhabditis elegans, a periodic calcium spike in a pacemaker cell initiates a calcium wave in the intestine. The calcium wave is followed by three muscle contractions that comprise the defecation motor program. Normal wave propagation requires the pannexin gap-junction subunit INX-16 at the interfaces of the intestinal cells. In the absence of this gap-junction subunit, calcium waves are frequently absent. The remaining waves are slow, initiate at abnormal locations, or travel in the opposite direction. Abnormal waves are associated with parallel effects in the first step of the motor program: The contractions of the overlying muscles fail to propagate beyond the pacemaker cell, are slow, initiate in abnormal locations, or are reversed. Moreover, the last two motor steps are predominantly absent. Finally, the absence of this gap-junction subunit also affects the reliability of the pacemaker cell; cycle timing is often irregular. These data demonstrate that pannexin gap junctions propagate calcium waves in the C. elegans intestine. The calcium waves instruct the motor steps and regulate the pacemaker cell's authority and reliability.

Intestinal calcium waves coordinate a behavioral motor program in C. elegans.

Periodic behavioral motor patterns are normally controlled by neural circuits, such as central pattern generators. We here report a novel mechanism of motor pattern generation by non-neural cells. The defecation motor program in Caenorhabditis elegans consists of three stereotyped motor steps with precise timing and this behavior has been studied as a model system of a ultradian biological clock [J.H. Thomas, Genetic analysis of defecation in C. elegans, Genetics 124 (1990) 855-872; D.W. Liu, J.H. Thomas, Regulation of a periodic motor program in C. elegans, J. Neurosci. 14 (1994) 1953-1962; K. Iwasaki, D.W. Liu, J.H. Thomas, Genes that control a temperature-compensated ultradian clock in Caenorhabditis elegans, Proc. Natl. Acad. Sci. USA 92 (1995), 10317-10321]. It was previously implied that the inositol-1,4,5-trisphosphate (IP3) receptor in the intestine was necessary for this periodic behavior [P. Dal Santo, M.A. Logan, A.D. Chisholm, E.M. Jorgensen, The inositol trisphosphate receptor regulates a 50s behavioral rhythm in C. elegans, Cell 98 (1999) 757-767]. Therefore, we developed a new assay system to study a relationship between this behavioral timing and intestinal Ca(2+) dynamics. Using this assay system, we found that the timing between the first and second motor steps is coordinated by intercellular Ca(2+)-wave propagation in the intestine. Lack of the Ca(2+)-wave propagation correlated with no coordination of the motor steps in the CaMKII mutant. Also, when the Ca(2+)-wave propagation was blocked by the IP3 receptor inhibitor heparin at the mid-intestine in wild type, the second/third motor steps were eliminated, which phenocopied ablation of the motor neurons AVL and DVB. These observations suggest that an intestinal Ca(2+)-wave propagation governs the timing of neural activities that controls specific behavioral patterns in C. elegans.

Differential regulation of TRPM channels governs electrolyte homeostasis in the C. elegans intestine.

The transient receptor potential (TRP) channels are implicated in various cellular processes, including sensory signal transduction and electrolyte homeostasis. We show here that the GTL-1 and GON-2 TRPM channels regulate electrolyte homeostasis in the C. elegans intestine. GON-2 is responsible for a large outwardly rectifying current of intestinal cells, and its activity is tightly regulated by intracellular Mg(2+) levels, while GTL-1 mainly contributes to appropriate Mg(2+) responsiveness of the outwardly rectifying current. We also used nickel cytotoxicity to study the function of these channels. Both GON-2 and GTL-1 are necessary for intestinal uptake of nickel, but GTL-1 is continuously active while GON-2 is inactivated at higher Mg(2+) levels. This type of differential regulation of intestinal electrolyte absorption ensures a constant supply of electrolytes through GTL-1, while occasional bursts of GON-2 activity allow rapid return to normal electrolyte concentrations following physiological perturbations.