Cody Gilleland

Large-scale in vivo femtosecond laser neurosurgery screen reveals small-molecule enhancer of regeneration

Discovery of molecular mechanisms and chemical compounds that enhance neuronal regeneration can lead to development of therapeutics to combat nervous system injuries and neurodegenerative diseases. By combining high-throughput microfluidics and femtosecond laser microsurgery, we demonstrate for the first time large-scale in vivo screens for identification of compounds that affect neurite regeneration. We performed thousands of microsurgeries at single-axon precision in the nematode Caenorhabditis elegans at a rate of 20 seconds per animal. Following surgeries, we exposed the animals to a hand-curated library of approximately one hundred small molecules and identified chemicals that significantly alter neurite regeneration. In particular, we found that the PKC kinase inhibitor staurosporine strongly modulates regeneration in a concentration- and neuronal type-specific manner. Two structurally unrelated PKC inhibitors produce similar effects. We further show that regeneration is significantly enhanced by the PKC activator prostratin.

Microfluidic immobilization of physiologically active Caenorhabditis elegans

We present a protocol for building and operating a microfluidic device for mechanical immobilization of Caenorhabditis elegans in its physiologically active state. The system can be used for in vivo imaging of dynamic cellular processes such as cell division and migration, degeneration, aging and regeneration, as well as for laser microsurgery, Ca2+ imaging and three-dimensional microscopy. The device linearly orients C. elegans, and then completely restrains its motion by pressing a flexible membrane against the animal. This technique does not involve any potentially harmful anesthetics, gases or cooling procedures. The system can be installed on any microscope and operated using only one syringe and one external valve, making it accessible to most laboratories. The device fabrication begins by patterning photoresist structures on silicon wafers, which are then used to mold features in elastomeric layers that are thermally bonded to form the device. The system can be assembled within 3 d.

Construction of a femtosecond laser microsurgery system

Femtosecond laser microsurgery is a powerful method for studying cellular function, neural circuits, neuronal injury and neuronal regeneration because of its capability to selectively ablate sub-micron targets in vitro and in vivo with minimal damage to the surrounding tissue. Here, we present a step-by-step protocol for constructing a femtosecond laser microsurgery setup for use with a widely available compound fluorescence microscope. The protocol begins with the assembly and alignment of beam-conditioning optics at the output of a femtosecond laser. Then a dichroic mount is assembled and installed to direct the laser beam into the objective lens of a standard inverted microscope. Finally, the laser is focused on the image plane of the microscope to allow simultaneous surgery and fluorescence imaging. We illustrate the use of this setup by presenting axotomy in Caenorhabditis elegans as an example. This protocol can be completed in 2 d.

Computer-Assisted Transgenesis of Caenorhabditis elegans for Deep Phenotyping

A major goal in the study of human diseases is to assign functions to genes or genetic variants. The model organism Caenorhabditis elegans provides a powerful tool because homologs of many human genes are identifiable, and large collections of genetic vectors and mutant strains are available. However, the delivery of such vector libraries into mutant strains remains a long-standing experimental bottleneck for phenotypic analysis. Here, we present a computer-assisted microinjection platform to streamline the production of transgenic C. elegans with multiple vectors for deep phenotyping. Briefly, animals are immobilized in a temperature-sensitive hydrogel using a standard multiwell platform. Microinjections are then performed under control of an automated microscope using precision robotics driven by customized computer vision algorithms. We demonstrate utility by phenotyping the morphology of 12 neuronal classes in six mutant backgrounds using combinations of neuron-type-specific fluorescent reporters. This technology can industrialize the assignment of in vivo gene function by enabling large-scale transgenic engineering.

Microfluidic in vivo screen identifies compounds enhancing neuronal regeneration.

Compound screening is a powerful tool to identify new therapeutic targets, drug leads, and elucidate the fundamental mechanisms of biological processes. We report here the results of the first in vivo small-molecule screens for compounds enhancing neuronal regeneration. These screens are enabled by the microfluidic devices we have developed for C. elegans. The devices enable rapid and repeatable animal immobilization which allows high-throughput and precise surgery. Following surgery, animals are exposed to the contents of a small-molecule library and assayed for neuronal regeneration. Using this screening method we have identified several compounds that enhance neural regeneration in vivo.

High-throughput in vivo genetic and drug screening using femtosecond laser nano-surgery, and microfluidics

We demonstrate microfluidic devices and imaging technologies for high-speed immobilization, cellular resolution imaging, and femtosecond laser micro-surgery of awake small-animals (C. elegans) for high-throughput in vivo genetic and drug screens on neural degeneration and regeneration.

High-throughput on-chip in vivo neural regeneration studies using femtosecond laser nano-surgery and microfluidics

In recent years, the advantages of using small invertebrate animals as model systems for human disease have become increasingly apparent and have resulted in three Nobel Prizes in medicine or chemistry during the last six years for studies conducted on the nematode Caenorhabditis elegans (C. elegans). The availability of a wide array of species-specific genetic techniques, along with the transparency of the worm and its ability to grow in minute volumes make C. elegans an extremely powerful model organism. We present a suite of technologies for complex high-throughput whole-animal genetic and drug screens. We demonstrate a high-speed microfluidic sorter that can isolate and immobilize C. elegans in a well-defined geometry, an integrated chip containing individually addressable screening chambers for incubation and exposure of individual animals to biochemical compounds, and a device for delivery of compound libraries in standard multiwell plates to microfluidic devices. The immobilization stability obtained by these devices is comparable to that of chemical anesthesia and the immobilization process does not affect lifespan, progeny production, or other aspects of animal health. The high-stability enables the use of a variety of key optical techniques. We use this to demonstrate femtosecond-laser nanosurgery and three-dimensional multiphoton microscopy. Used alone or in various combinations these devices facilitate a variety of high-throughput assays using whole animals, including mutagenesis and RNAi and drug screens at subcellular resolution, as well as high-throughput high-precision manipulations such as femtosecond-laser nanosurgery for large-scale in vivo neural degeneration and regeneration studies.