Mondal S et al. (2011) Traffic "Imaging in vivo neuronal transport in genetic model organisms using microfluidic devices."

Rau K, Venkataraman V, Mondal S, Ahlawat S, Koushika SP

[Traffic, 2011]

Microfluidic devices have been developed for imaging behavior and various cellular processes in Caenorhabditis elegans, but not subcellular processes requiring high spatial resolution. In neurons, essential processes such as axonal, dendritic, intraflagellar and other long-distance transport can be studied by acquiring fast time-lapse images of green fluorescent protein (GFP)-tagged moving cargo. We have achieved two important goals in such in vivo studies namely, imaging several transport processes in unanesthetized intact animals and imaging very early developmental stages. We describe a microfluidic device for immobilizing C. elegans and Drosophila larvae that allows imaging without anesthetics or dissection. We observed that for certain neuronal cargoes in C. elegans, anesthetics have significant and sometimes unexpected effects on the flux. Further, imaging the transport of certain cargo in early developmental stages was possible only in the microfluidic device. Using our device we observed an increase in anterograde synaptic vesicle transport during development corresponding with synaptic growth. We also imaged Q neuroblast divisions and mitochondrial transport during early developmental stages of C. elegans and Drosophila, respectively. Our simple microfluidic device offers a useful means to image high-resolution subcellular processes in C. elegans and Drosophila and can be readily adapted to other transparent or translucent organisms.

Mondal S et al. (2012) J Vis Exp "Simple microfluidic devices for in vivo imaging of C. elegans, Drosophila and zebrafish."

Mondal S, Koushika SP, Ahlawat S

[J Vis Exp, 2012]

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques. Microfluidic devices have been used for sub-cellular imaging, in vivo laser microsurgery and cellular imaging. In vivo imaging requires immobilization of organisms. This has been achieved using suction, tapered channels, deformable membranes, suction with additional cooling anesthetic gas, temperature sensitive gels, cyanoacrylate glue and anesthetics such as levamisole. Commonly used anesthetics influence synaptic transmission and are known to have detrimental effects on sub-cellular neuronal transport. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from -10 m to -800 m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min. We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F). Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in -30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Mondal S et al. (2014) Methods Mol Biol "Microfluidic devices for imaging trafficking events in vivo using genetic model organisms."

Koushika SP, Mondal S

[Methods Mol Biol, 2014]

Miniature devices are powerful new tools that can be used to address multiple questions in biology especially in investigating an individual cell or organism. The primary step forward has been the ease of soft lithography fabrication which has allowed researchers from different disciplines, with incomplete technical knowledge, to develop and use new devices for their own research problems. In this chapter, we describe a simple fabrication process that will allow investigators to make microfluidic devices for in vivo imaging studies using genetic model organisms such as C. elegans, Drosophila larvae, and zebrafish larvae. This microfluidic technology enables detailed studies on multiple cellular and subcellular phenomena including intracellular vesicle trafficking in living organisms over different developmental stages in an anesthetic free environment.

Mondal S et al. (2016) Nat Commun "Large-scale microfluidics providing high-resolution and high-throughput screening of Caenorhabditis elegans poly-glutamine aggregation model."

Ghorashian N, Martin C, Mondal S, Gokce SK, Ben-Yakar A, Hegarty E

[Nat Commun, 2016]

Next generation drug screening could benefit greatly from in vivo studies, using small animal models such as Caenorhabditis elegans for hit identification and lead optimization. Current in vivo assays can operate either at low throughput with high resolution or with low resolution at high throughput. To enable both high-throughput and high-resolution imaging of C. elegans, we developed an automated microfluidic platform. This platform can image 15 z-stacks of 4,000 C. elegans from 96 different populations using a large-scale chip with a micron resolution in 16min. Using this platform, we screened 100,000 animals of the poly-glutamine aggregation model on 25 chips. We tested the efficacy of 1,000 FDA-approved drugs in improving the aggregation phenotype of the model and identified four confirmed hits. This robust platform now enables high-content screening of various C. elegans disease models at the speed and cost of in vitro cell-based assays.

Mondal, S. et al. (2017) International Worm Meeting "High-throughput screening technology identifies dronedarone HCl as proteostasis regulator in C. elegans polyglutamine aggregation models."

Ghorashian, N., Gokce, S.K., Mondal, S., Ben-Yakar, A., Hegarty, E., Martin, C.

[International Worm Meeting, 2017]

The nematode Caenorhabditis elegans has been proved to be a useful in vivo model for next generation drug screening to identify new hits and optimize novel leads. Current in vivo assays can operate either at low throughput with high resolution or with low resolution at high throughput. To enable both high-throughput and high-resolution imaging of C. elegans models labeled with different fluorescent reporters, we developed an automated microfluidic platform. The platform includes a large-scale microfluidic chip with 96-well designed in standard microtiter plate format and densely packed trapping channels in each well. The channels are uniquely designed to immobilize approximately 4,000 animals simultaneously in 3 min in a lateral orientation. The automated imaging platform can image 15 z-stack images of all trapped animals, capturing their whole volume with a micron resolution in less than 16 min. An automated graphical user interface (GUI) loads all the images, identifies the single animal and perform image processing steps to identify the aggregates present in the body wall muscle cells of our C. elegans polyglutamine aggregation models. Using this platform, we screened ~100,000 animals of the polyglutamine aggregation model, with 35 glutamine repeats, using a total number of 25 chips operated with a Z'-factor of 0.8. We tested the efficacy of ~1,000 FDA approved drugs in improving the aggregation phenotype of the model and identified 4 confirmed hits, one of which has a strong dose-response. The confirmed hit was verified on a different strain with 40 polyglutamine repeat length, showing a protein aggregation in the much earlier stage of the development, using our microfluidic platform in a dose-dependent manner. This robust platform now enables high-content screening of various C. elegans disease models at the speed and cost of in vitro cell-based assays.

Mondal, S. et al. (2019) International Worm Meeting "Probabilistic addition of mitochondria in the growing touch neuronal process identified using a long-term growth and imaging microfluidic device."

Koushika, S.P., Mondal, S., Awasthi, A., Sure, G.P.R., Dubey, J.

[International Worm Meeting, 2019]

The long cellular architecture of neurons requires regulation in part through transport and anchoring events to distribute intracellular organelles. Mitochondria are one such organelle that are present at a constant density in touch neurons. As the neuron grows with increase in animal length, mitochondria are added at the rate of ~0.4 per hour to to result in the density of ~5/100 ?m in all adult animals. To identify how mitochondria are added along the neuron we developed a microfluidic device to feed and immobilize C. elegans for high-resolution imaging over a period of several days. Repeated sub-cellular imaging of an identified touch neuron in a given animal, over 36 hours in our microfluidic device, shows no preferential location for mitochondrial addition. New mitochondria are added anywhere along the neuronal process when the average separation between the two pre-existing mitochondria crosses the threshold of 24 micrometers. Mitochondria along the neuronal process show turnover and new mitochondria may arise due to entry from the cell body and from fission of moving mitochondria in the neuronal process. Using our long-term growth and imaging device we have identified a how a slow cell biological process occurs in individual neurons in identified animals without population averaging.

Mondal S et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Imaging in vivo axonal transport in C. elegans using microfluidic devices"

Venkataraman V, Ahlawat S, Mondal S, Rau K, Kouhsika S P

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Microfluidic devices for C. elegans have been developed for imaging various cellular or behavioural processes. However they have not been utilized for studying high resolution sub-cellular process. Axonal transport is an essential process where moving cargo transport can be imaged using fast acquisition time lapse images of GFP tagged molecules in vivo. We have achieved two important goals in such in vivo studies namely, imaging unanesthetized intact animals and imaging very early developmental stages. We describe a PDMS microfluidic device for immobilizing C. elegans that allows imaging without the use of anesthetics. We observed that anesthetics have significant and in some cases unexpected effects on the flux of cargo in the axon, making it a less than optimal method of imaging axonal transport. Further, imaging cargo transport in early developmental stages was possible only in the microfluidic device. Using our device we were able to observe increase in anterograde synaptic vesicle transport during synaptic growth corresponding with an increase in vesicle deposition at synaptic regions. In summary our simple microfluidic device offers an improved method to image any high resolution sub-cellular process in C. elegans. Our device principles can also be readily adapted to other transparent or translucent model organisms.

Mondal, S. et al. (2019) International Worm Meeting "A whole-animal confocal imaging platform shows oscillating motor neuron activities in the basal motor neuron circuit."

Ben-Yakar, A., Zimmer, M., Kato, S., Mondal, S., Martin, C., Hegarty, E., ZHAO, P., Duplissis, A., Kim, K.H.

[International Worm Meeting, 2019]

One of the many complex behaviors is the locomotion of living creatures that are controlled by a well-coordinated neural network and the neuromuscular junctions. It has been difficult to probe and understand this complex behavior in the larger animals. The simplicity of the invertebrate neuronal system has helped to identify system-level information to understand the biological process underlying how complex neuronal networks operate. In order to study latent motor neuron activities, we capture all the motor neurons present in the ventral cord of a larval 4 stage C. elegans. The animal is immobilized inside a meander-shaped microfluidic channel to mimic its natural body posture during crawling. A custom-designed confocal system with a flexible field of view is used to image the entire animal within 630 × 160 mum2 and using 430 nm optical resolutions. We characterized the basal activities of the entire motor neuron circuit in the anesthetic-free animal to identify oscillatory neuron responses using imaging rate of >5 volumes per second. To autonomously extract each neuron's signal, we denoise each volume via multiscale analysis and identify unique neurons based on a set of pixel-wise statistical and morphological features. An interactive software allows for human input and customizes neuron identification to correct for the errors made during automated analysis before each neural trace is extracted for further analysis. We observed sparse latent signaling patterns in the C. elegans motor activities that can be described well by a low-dimensional space, within some bound of error, to a high degree of precision.

Mondal S et al. (2018) ACS Chem Neurosci "2R/Tmem97 binding ligands reduce age-dependent neurodegeneration in C. elegans SC_APP model using a high-content microfluidic screening platform."

Martin SF, Martin C, Ben-Yakar A, Satarasinghe PN, Hegarty E, Mondal S, Gokce SK, Sahn JJ, Iyer S, Sae-Lee W, Hodges T, Scott LL, Ghorashian N, Pierce JT

[ACS Chem Neurosci, 2018]

The nematode Caenorhabditis elegans, with tractable genetics and a well-defined nervous system, provides a unique whole-animal model system to identify novel drug targets and therapies for neurodegenerative diseases. Large-scale drug or target screens in models that recapitulate the subtle age- and cell-specific aspects of neurodegenerative diseases are limited by a technological requirement for high-throughput analysis of neuronal morphology. Recently we developed a model of amyloid precursor protein-induced neurodegeneration that exhibits progressive degeneration of select cholinergic neurons. Our previous work with this model suggests that small molecule ligands of the sigma 2 receptor (2R), which was recently cloned and identified as transmembrane protein 97 (TMEM97), are neuroprotective. To determine structure-activity relationships for unexplored chemical space in our 2R/Tmem97 ligand collection, we developed an in vivo high-content screening (HCS) assay to identify potential drug leads. The HCS assay uses our recently developed large-scale microfluidic immobilization chip and automated imaging platform. We discovered norbenzomorphans that reduced neurodegeneration in our C. elegans model, including two compounds that demonstrated significant neuroprotective activity at multiple doses. These findings provide further evidence that 2R/Tmem97-binding norbenzomorphans may represent a new drug class for treating neurodegenerative diseases.

Dubey J et al. (2022) J Vis Exp "A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans."

Mondal S, Koushika SP, Dubey J

[J Vis Exp, 2022]

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding these biological processes often requires long-term and repeated imaging of the same animal. Long recovery times associated with conventional immobilization methods done on agar pads have detrimental effects on animal health making it inappropriate to repeatedly image the same animal over long periods of time. This paper describes a microfluidic chip design, fabrication method, on-chip C. elegans culturing protocol, and three examples of long-term imaging to study developmental processes in individual animals. The chip, fabricated with polydimethylsiloxane and bonded on a cover glass, immobilizes animals on a glass substrate using an elastomeric membrane that is deflected using nitrogen gas. Complete immobilization of C. elegans enables robust time-lapse imaging of cellular and sub-cellular events in an anesthetic-free manner. A channel geometry with a large cross-section allows the animal to move freely within two partially sealed isolation membranes permitting growth in the channel with a continuous food supply. Using this simple chip, imaging of developmental phenomena such as neuronal process growth, vulval development, and dendritic arborization in the PVD sensory neurons, as the animal grows inside the channel, can be performed. The long-term growth and imaging chip operates with a single pressure line, no external valves, inexpensive fluidic consumables, and utilizes standard worm handling protocols that can easily be adapted by other laboratories using C. elegans.