undergraduate research-final

1Christian Jaques Hissom

Tuszynski lab, Center for Neuronal [email protected] (805)-637-5653

I. TitleInvestigating the widespread collateralization of corticospinal neurons using a two-viral vector tracing strategy in the adult mammalian motor system.

II. Abstract of Project Corticalspinal neurons in the motor cortex with descending projections to the spinal cord play a critical role in motor movement. While prior studies have observed the presence of corticalspinal tract (CST) collaterals, the extent of the CST’s branching pattern is largely unknown. The length of the axon challenges traditional tracing techniques, which rely on diffusible dyes. This impedes successful CST collateral labeling. The goal of the present study is to define CST collaterals in the rat using novel viral strategies. Evidence for widespread collateralization will challenge the longstanding views regarding the functional role of this descending system in motor control.

III.Detailed description and timeline Theory: Surgical delivery of a two-viral vector strategy is the fist part of this project and will be completed before the beginning of summer. First, a Cre-expressing virus will be injected into the spinal cord to retrogradely infect corticospinal neurons in the brain. Next, a Cre-dependent virus expressing a membrane targeted fluorescent tracer (AAV-FLEX-ArchT-tdTomato) will be injected into the motor cortex. Only cells infected with both viruses will express the membrane targeted tracer. In this manner, this two viral vector strategy provides the specificity for and enhances the efficacy of CST neuronal tracing. The lab has developed and tested the proposed viral tracing strategy in multiple systems and we have recently demonstrated that we can successfully target the FLEX-ArchT-tdTomato tracer to corticospinal neurons. Working alongside this lab for nearly two years, I have learned how to carry out the necessary procedures in order to complete the experiment. These procedures include: surgery, perfusion, tissue cutting and processing, histology, and microscopy. I will obtain the necessary training for the production of a 3d model representation to be used for all subsequent research akin to corticospinal neurons. All procedures and animal care adhered to American Association for the Accreditation of laboratory Animal Care, Society for Neuroscience, and institutional guidelines for experimental animal health, safety, and comfort.Perfuse rats: More than 20 male F344 rats weighing between 90-250g will be perfused 3 weeks after receiving the stereotaxic injections of viral vectors. Perfusion is achieved by surgically infusing the tissue with paraformaldehyde (PFA). Perfusion entails an incision into the left ventricle and clamping of the catheter into the aorta. A subsequent incision of the right atrium creates a fixed system for delivery of PFA by way of the blood stream to every tissue. Post PFA infusion, the scull and spine is removed and the brain is extracted using rongeur clippers. The extracted brain will be incubated in PFA for an additional 3 days and then transferred to .5% sucrose solution. Afterwards, the cord will be dissected from the spine and transferred to a sucrose solution.

mailto:[email protected]



Cut tissue: The tissue will be cut at 40 µm by blocking the brain on an ice stage standing vertical from occipital to frontal lobe. Coronal sections will be collected and organized for the brain and spinal cord. Stain tissue: Tissue staining will be achieved by green fluorescent protein, or red fluorescent protein staining using immunohistochemistry protocol for amplification of the signal. A subset of the tissue will also be processed using a nissl stain protocol to provide a secondary visualization technique for anatomical elucidation of coticostpinal tract collaterals. Image stained sections: GFP/RFP Stained sections will be mounted on subbed microscope slides and analyze using modified software MetaXpress; a 96-well type microscope devise. Sections will be recorded and layered by the computer software providing the data that will late be used to create a dimensional reconstruction of CST collateral projections.Reconstruct CST projections: A three-dimensional model and blueprint of CST collaterals will be produced after using MetaXpress to compile all collected tissue data. These data will be paired with subsequent anatomical elucidation of collateral and corresponding brain regions. This project will provide a model for future studies akin to motor function and strengthen our understanding of CST collateral communication. IRB: An IRB will not be needed for this project.



June 15th – August 28th

Objective Tasks Hours/Week

Week 0Perfuse rats1. Animals will be

perfused2. Brains and spinal

cords will be dissected.

<20 rats will be perfused with paraformaldehyde.

Extracted tissue will be incubated in paraformaldehyde for up to 3 days and then transferred to sucrose solution prior to cord dissection.

40-60 hours/ week are expected

for the timely

completion of each

experimental procedure

Week 1

Cut tissue1. Brain sections2. Spinal cord sections

Extracted brain post perfusion will be sectioned in the coronal plane at 40 µm

Extracted spinal cords post perfusion will also be sectioned in the coronal plane at 40 µm

Sections will be collected from all four segments of the spinal cord: cervical, thoracic, lumbar, and sacral, C1-L6 respectively.

Week 2

Week 3

Stain tissue1. Primary antibody2. Secondary antibody3. Biotynyl tyramide

amplification4. DAB blocking

Sections, spaced 240 µm apart, will be stained using an antibody to td-Tomato.

Tissue will first be tagged using a primary antibody.

Between 1-3 days of incubation are needed prior to the addition of the secondary antibody.

Week 4 Processed tissue will be washed first with TBS and prepared for addition of secondary antibody.

Secondary antibody will require up to 3 hours of incubation for each set of collected brain and corresponding spinal cord.

Biotynyl tyramide incubation for 30 minutes will amplify the antibody signal and improve efficacy of subsequent staining.

Subsequent td-Tomato reagent staining for visualization of ARchTd-Tomato will reveal the full extent or corticospinal tract axonal collaterals.

Week 5 Image stained sections1. Microscope slide

mounting2. MetaXpress and

microscopy

Stained tissue will be organized and arranged on subbed microscope slides according to brain and spinal cord regions.

Week 6 Stained sections will be imaged using MetaXpress imaging program.Week 7

Week 8 Reconstruct CST projections1. Reassemble sections2. Anatomical

identification of collaterals

All sections will then be reassembled to create a 3D atlas of corticospinal projections

Week 9 Brain regions innervated by corticospinal terminals will be identified using the Paxinos and Watson Atlas of the Rat Brain.

Week 10

IV. Personal statement



As a child I always knew my grandmother as a paraplegic. Bound to a wheelchair and with limited movement, she wore a vibrant smile and held static personality that adequately concealed her discomfort and pain. My grandfather did everything to improve her life. Together they endured the hardship that her debilitation provided. Everything came to an end after my grandfather was diagnosed with cancer. A few months after he passed away, my grandmother's vibrant smile no longer concealed the pain. I was surprised that a woman’s life could fade to a broken heart and I felt disappointed by modern medicine’s inability to save their lives. In my youth I wanted to be an innovator and an engineer. As I grew older I decided to apply these mentalities to medicine and the field of neuroscience.

My career as a scientist began as a lab technician with prof. Leigh Dicks in the physiology department lab of Santa Barbara community college. I remember clearly my first day at work: cabinets filled with beakers and glassware, chemical and scales, that all bearded the sharp realization that this was my first step into the beginning of a thrilling journey.

I spent my summer involved in two summer research programs as I transferred from community college to UCSD. First, I worked with Dr. Blake Gillespie on the kinematics of protein folding as at CSUCI through Project ACCESO. The internship ended with my first poster presentation which brought to my attention my passion for scientific demonstrations and strengthened my ambition to become a researcher. That week, I learned I had been accepted into the MARC program. I explored several labs before I found Dr. Mark Tuszynski’s lab. Their research entailed the concepts of brain plasticity and spinal cord recovery that most captivated my curiosity. I met with Dr. James Conner, project scientist, and learned about the innovative viral approaches they employed for systems research akin to motor movement. I was astounded by the specificity of this research and inspired by the ability to improve modern medicine. That summer I began working with Dr. Conner investigating corticospinal and thalamocortical neurons.

For the past two years I have worked alongside Dr. Conner, exploring the function of the damaged neurons that restricted my grandmother’s movement. Now I have been granted the opportunity to be part of a grand contribution to this field by elucidating the branching patterns of corticospinal neurons. This project will strengthen the field’s understanding by providing a blueprint of the corticospinal tract for subsequent research; research that I hope to pursue. My part in this project will strengthen my existing skills with surgical and histological procedures. I will also gain valuable analytical and computer modeling training. I hope to someday have my own lab and, like Dr. Tuszynski and Dr. Conner, produce outstanding research that will benefit the well-being and lives of those afflicted by ailments of the central nervous system.

undergraduate research-final

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