Arisaka, Katsushi et al. (2017) International Worm Meeting "Dynamic Connectome of C. elegans Neural Networks for Spatial Navigation based on Central Pattern Generator."

Lee, Se-Young (Myki), Arisaka, Katsushi, Jin, Suying, Hydari, Syed (Bashir)

[International Worm Meeting, 2017]

Neural networks have arisen in Animalia over vast evolutionary time spans as a means to sense environmental stimuli and take effective navigational action. However, a critical issue lies in the fact that sensory neurons are only able to detect temporal variations caused by external stimuli at the specific location of the receptors, which contain no inherent spatial information of external environment. This raises the question of how such a collection of signals in time is converted into a sufficient understanding of the surrounding space to inform an appropriate reaction. There must be a mechanism for the conversion of signals in time into a distribution in space, and we argue this "conversion factor" must be supplied by the animal's own motion. We propose a novel concept that such time-to-space conversion can be effectively achieved by utilizing a periodic rhythm of brain activity, Central Pattern Generator (CPG) as a universal clock to synchronize neural networks. This hypothesis has been applied to the C. elegans nervous system to establish the "dynamic connectome" which builds on the well established static connectome by predicting the dynamic function of these circuits especially as they pertain to sensorimotor integration and consequent perception of space. We identify particular groups of motor neurons exhibiting cross inhibitory connections as the prime candidates to be CPGs. We predict the sequence of of neural activity for the process of sensory signals arriving via interneurons to the motor neurons while spatial context is provided by the current state of the rhythmic activity of the proposed CPG motor neurons separately controlling body and head orientation. Several experimental evidences supporting this concept will be presented.

Jin, Suying et al. (2017) International Worm Meeting "An adaptable machine vision package for whole brain cellular resolution neural activity analysis in freely navigating Caenorhabditis Elegans."

Arisaka, Katsushi, Jin, Suying

[International Worm Meeting, 2017]

Recent advances in bio-imaging have made large scale recordings of neural activity at cellular resolution possible; providing the unprecedented opportunity to observe the dynamic activity of the entire nervous system in Caenorhabditis elegans. While software to analyze stationary data taken on immobilized animals is both publicly available and straightforward enough to develop, imaging immobilized animals has clear scientific limitations. In order to study the full scope of the complex transformation of sensory inputs to appropriate behaviors, the sample must be allowed to freely behave under a variety of controlled stimulations, at which point the challenge of detecting of moving objects is encountered?an active field in computer vision research. We have developed a lightweight software package capable of detecting and quantifying calcium dynamics in freely navigating C. elegans. A frame-by-frame static detection of intensity maxima is performed on a rapidly scanned volumetric dataset, followed by a variety of inference methods to establish the continuous identities of neurons and calculate their respective activity (measured by delta F/F0). Data taken with strain QW1217 was used for the development of the software, however only the dynamic GCaMP6 channel was used for the analysis and static nuclear markers such as RFP are not required for neuron detection. We also make no presumption on the hardware setup of the user with the hopes of increasing accessibility, compatibility and applicability for any future neurodynamic investigations. Experimental results have shown sufficient neural identification and tracing with minimal manual proofreading, so long as the volumetric acquisition rate is high compared to the worm's translation through the field of view, such that the movement of a typical neuron will not exceed its diameter in the time between image volumes.

Lyashenko, Alexey et al. (2015) International Worm Meeting "Line scanning fluorescent confocal microscope for a 3D multi-neuron imaging of C. elegans."

Polanco, Edward, Lyashenko, Alexey, Arisaka, Katsushi

[International Worm Meeting, 2015]

Conventional confocal microscope uses a physical aperture to reduce the amount of out of focus light to the image sensor. We developed a line scanning confocal microscope that utilizes a software controlled rolling shutter on a CMOS camera for a high-speed 3D volume imaging of dozens of active neurons. The microscope setup allows for a real time worm tracking for freely navigating C. elegans under a localized external stimulation for phototaxis and thermotaxis. An external photo stimulation for optogenetics was also realized.

Carmona, Christopher et al. (2015) International Worm Meeting "Understanding Neurodynamics Through Light Field Microscopy."

Arisaka, Katsushi, Polanco, Edward, Carmona, Christopher, Madruga, Blake, Hammond, Addam

[International Worm Meeting, 2015]

Light field microscopy is a new technique that allows for quick volumetric imaging of fluorescent specimens. It utilizes a microlens array (MLA) as a key optical component that produces a light field and trades spatial resolution against angular resolution or axial resolution. The MLA is a matrix of lenses with diameters of 130mum that each resolve a visual perspective of a specimen being imaged at relative distances from the native object plane. As a result, recorded light-fields can be computationally reconstructed into full volumes. The volume reconstruction is formulated as an inverse linear transformation that is modeled using wave optics theory. Here, an epifluorescence light field microscope is designed and configured in order to resolve the neural activity of C. elegans during real-time inculcated behavior. The theoretical limits of the microscope's lateral resolution in relation to optical design choices are discussed and compared with experimental results. The primary focus of this investigation is the utilization of light field microscopy in conjunction with computationally intensive image processing methods as a useful tool for analyzing the behavior and corresponding brain activity of C. elegans. Light field microcopy has the potential to offer real-time 3-D video data of the unrestrained behavior of C. elegans.

Madruga, Blake et al. (2017) International Worm Meeting "Development of a SLM-based sheet illumination microscope for rapid, large-scale, video-rate 3-D neurodynamic observation in C. elegans."

Mendoza, Steve, Madruga, Blake, Carmona, Javier, Thatcher, Joe, Arisaka, Katsushi

[International Worm Meeting, 2017]

Sheet Illumination microscopy has made a large impact on the microscopy community due to its many inherent advantages. Increased photonic efficiency allows for lower power light sources, which in turn reduce phototoxic damage to the sample while providing an increased signal to noise ratio. To take advantage of such technique, a type of phase modulator, known as a Spatial Light Modulator (SLM) is used to generate a deep-penetrating, extremely long and narrow Bessel beam interference pattern. Through the use of an SLM, one can easily modulate the phase characteristics of an illumination beam in real time. This property enables greater flexibility and aberration compensation at the sample. The Bessel beams are generated through a bitmap image of the beam's modulation transfer function, which is then displayed on the SLM with a prism phase rotation. This provides a much longer region of micron-order uniformity along the beam axis compared to conventional Gaussian geometries, while shifting the modulated beam away from the higher diffraction orders. The beams are then mapped onto the readout of two scientific CMOS cameras for rapid multi-channel imaging. A piezoelectric objective collar is used to enable rapid z-scanning, thereby creating 4D image volumes with adequate time resolution to characterize and observe active neural dynamics in C. elegans. A long working distance, high numerical aperture (NA), refractive-corrected objective lens is used to study neurobiology and participate in ratiometric calcium imaging. Tools of such flexibility will enable the study of whole-brain-scale neuronal activity and structure under various controlled conditions in C. elegans, over a variety of temporal and spatial scales.

Jiang, Karen et al. (2015) International Worm Meeting "Sensorimotor signal transmission through AIY interneuron in Caenorhabditis elegans during isothermal tracking."

Ling, Linsay, Mendoza, Steve, Sherry, Tim, Nowak, Nathaniel, Arisaka, Katsushi, Jiang, Karen, Haller, Leonard

[International Worm Meeting, 2015]

Perception and navigation through space require accurate translation and transmission of sensory input to motor output. On a linear temperature gradient, Caenorhabditis elegans demonstrate a distinct behavioral phenotype in which they frequently travel along isotherms, maintaining sensitivity within 0.05 degC. This isothermal attractor state is correlated with movement at a constant and maximal velocity. We investigate how AIY, a first layer interneuron and postsynaptic partner to AFD thermosensory neuron, is able to integrate thermal information to return specific well-defined behavioral phenotypes. Prior observations of neural activity in vivo involve partial paralysis or constraint of the worm while stimuli is applied. Other systems circumvent this limitation by re-centering the stage; this generates an external force during stage acceleration introducing another stimulus. We overcome these two primary obstacles through the implementation of a novel automated worm-tracking epi-fluorescent microscope. The three-camera microscope system mounted on a movable XY stage captures dynamic Ca+2 signals in Cameleon-labeled neurons while the nematode navigates unconstrained along the temperature gradient. Implementing this set up, we observed that the greatest temperature difference occurs between the extremes of the head movement while along isotherms which phase lock with fluorescence response in AIY. The steady Ca+2 waveform in AIY suppresses reversals and maintains high speeds to downstream motor circuitry. .

Madruga, Blake et al. (2015) International Worm Meeting "Development of a Bessel Beam Sheet Illumination Microscope for Large-Scale 3-D Neural Dynamics in C. elegans."

Chin, Paul, Arisaka, Katsushi, Madruga, Blake, Carmona, Christopher, Sherry, Tim, Mendoza, Steve, Chen, William, Polanco, Edward

[International Worm Meeting, 2015]

Sheet Illumination has recently amassed a lot of attention as a technique, due to many benefits over standard microscopy methods. Decreased phototoxicity, increased signal to noise ratio, and higher photonic efficiency are only a few of the reasons why many researchers are beginning to answer sensitive scientific questions with sheet illumination microscopy. Due to the unique properties and biological characteristics of C. elegans, access to sheet illumination microscopy is limited, costly, and difficult to utilize. The purpose of designing such a device is to bring the proven benefits of sheet illumination to the C. elegans community, in a intuitive, purposefully-designed manner. A type of phase modulator, known as a Spatial Light Modulator (SLM) is used in this case to generate a specific bessel-beam pattern. Through the use of an SLM, one can easily modulate multiple characteristics of the illuminative beam in real time, enabling great flexibility, ensuring high resolution across multiple scientific applications.Additionally, the use of a piezoelectric objective collar allows the rapid capturing of three dimensional volumes, enabling researchers to examine the dynamics of many neurons in space and time, at sub-micron axial resolution. Such tools will gain access to the observation of large-scale, three dimensional neuronal activity under controlled or experimental conditions in C. elegans, leading to potential scientific discoveries in the future.

Carmona, Javier et al. (2017) International Worm Meeting "2D worm tracking line-confocal microscope with virtual reality."

Wang, Charles, Carmona, Javier, Baldo, Anthony, Madruga, Blake, Mendoza, Steve, Jin, Suying, Arisaka, Katsushi, Liu, Larry, Thatcher, Joseph

[International Worm Meeting, 2017]

Calcium dynamic imaging and free motion tracking coupled with external stimulations allow for in depth analysis of C. elegans behavior. We have developed an integrated platform for monitoring and controlling C. elegans under a variety of external stimulations, including thermal, electrical, and photo stimuli. This innovative platform combines rapid volumetric (20 volume/s) diffraction limited dual line-confocal microscopy (0.5 um x 1 um x 5 um voxel) to determine the neural pathways different external stimuli induce, while tracking worm's two dimensional motion. Never before has dynamic signal propagation, from neuron to neuron, been observed for C. elegans in free motion at such high volume scanning rate. External stimuli are computer controlled with < 10 ms resolution for precise spatio-temporal synchronization with free motion behavior and whole-brain calcium dynamics. Physical linear and circular thermal gradients were implemented using customized temperature plates with thermal fluctuations of less than 0.05 deg C. In addition, thermal stimulation was applied via a 1490 nm infrared laser to create virtual temperature conditions, synchronized with head motion. Infrared laser stimulation allows C. elegans' thermoreceptor (AFD neuron) to perceive temperature fluctuations exclusively in the time domain, thereby allowing for the complete virtual manipulation of the nematode's thermal environment. Electrical responses were induced using a technique that involves applying a linear or spatially alternating electrical field through a gelatin sample with fields ranging from 4 to 14 V/cm. Photon stimulation was implemented using a 405 nm laser with intensities ranging from 0 to 10 mW/mm^2. Volumetric Calcium imaging of QW1217 has also allowed for the complete mapping of the neurons responsible for each of the aforementioned stimuli. The microscope and software accommodate multiple simultaneous stimuli applications, such as electrical and photon simulations. Also tested were the neural pathway differences between infrared and photo avoidance behavior due to their very similar behavioral responses.

Mendoza, Steve et al. (2015) International Worm Meeting "A Novel Worm Tracking Calcium Imaging System Utilizing a Mobile Microscope."

Sherry, Tim, Lam, Brian, Kao, Michelle, Nowak, Nate, Mendoza, Steve, Kim, Taejoon, Arisaka, Katsushi, Madruga, Blake, Jiang, Karen

[International Worm Meeting, 2015]

Worm tracking of freely moving worms is essential to study the connection between behavior and neural activity. However, these microscopes are often limited in their ability to track worms under various behavioral simulations. We present a fluorescence worm tracking microscope that has an open geometry and thus can track worms even if the behavioral platform is difficult or impossible to move via a motorized stage. As opposed to other automated worm tracking systems, our microscope is fully mobile-where all the optical components are mounted on top of a motorized xy stage-while the sample stage where C. elegans rests is stationary. This platform allows for ratiometric calcium imaging while also tracking a dark field worm image for behavioral analysis, running at 15 frames per second. The current configuration has three cameras, two for each of the YFP and CFP channels, and a dark field image showing the worm body, under a 10x magnification; the microscope can also be adjusted to image at 20x. Being able to track freely moving worms without moving the sample stage, allows our microscope to perform worm tracking in experimental conditions that similar systems have not been able to achieve. In addition, since the sample is stationary, we also avoid introducing confounding effects on the worms due to stage acceleration. To test this novel hardware, we run a thermotaxis experiment tracking a worm without moving the temperature platform. Our worm was labeled with AIY::CAM and we find a correlation between the AIY activity and the temperature of the head location during isothermal behavior. Our methodology could also apply to other behavioral experiments where an external stimulus would be hard to move via a motorized stage, such as an electrotaxis experiment.

Thatcher, Joseph et al. (2017) International Worm Meeting "Development of a three dimensional fluorescence tracking microscope for the motional analysis and behavioral stimulation of C. elegans."

Lam, Brian, Madruga, Blake, Carmona, Javier, Shrestha, Ahis, Jin, Suying, Mendoza, Steve, Thatcher, Joseph, Arisaka, Katsushi, Niaki, Shayan

[International Worm Meeting, 2017]

Extensive advances have been made in understanding the behavior of C. elegans in two dimensional environments. However, they impose substantial constraints on the worm's motion and ultimately restrict the set of possible natural behavioral states it can demonstrate. Addressing limitations encountered by previous efforts in three dimensional imaging, we designed and built a microscope capable of tracking the motion of C. elegans via a set of motorized stages while navigating freely within a sample volume. The variation of gelatin concentration (1% - 4%) and the utilization of temporally controlled ultraviolet photo-stimulation (405 nm) were also incorporated into the system. The addition of a refractive index mismatch correction chamber and fluorescence detection enable novel opportunities for observation and categorization of motion. Preliminary data of photoavoidance response in three dimensions was acquired and demonstrates the added complexity present in an unconstrained response. A novel use of fluorescence enables the identification of C. elegans' absolute orientation with respect to the ventral nerve cord. A model of motion based on sinusoidal wave propagation was applied to C. elegans' forward locomotion, thereby categorizing a set of three dimensional body states inhabited. From this analysis, we have identified three distinct motional states: one of which is sinusoidal in the worm's ventrodorsal plane, another which is sinusoidal in their lateral plane, and a final state that is helical in shape. Fitting this parametric model allows the extraction of a variety of wave-based parameters including wavelength, frequency, wave speed and phase difference which may then be correlated with other dynamic quantities and gelatin concentrations. Namely, the phase difference acts as a direct indicator of the degree to which the worm's posture is planar or helical, allowing the ability to parameterize its general motional form with a single number. Furthermore, from pre-existing, established data of the C. elegans' connectome, we hypothesize a neuronal mechanism for rhythmic signal generation based on the SMD motor neurons which predicts the motional states observed.