1. 1
ChristianJaquesHissom
Tuszynski lab,Center forNeuronal Repair
Chissom@ucsd.edu (805)-637-5653
I. Title
Investigating 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.

2. 2
ChristianJaquesHissom
Tuszynski lab,Center forNeuronal Repair
Chissom@ucsd.edu (805)-637-5653
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.

3. 3
ChristianJaquesHissom
Tuszynski lab,Center forNeuronal Repair
Chissom@ucsd.edu (805)-637-5653
June 15th
–
August 28th
Objective Tasks Hours/Week
Week 0
Perfuse rats
1. Animals will be
perfused
2. 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 priorto cord
dissection.
40-60
hours/ week
are expected
for the
timely
completion
of each
experimenta
l procedure
Week 1
Cut tissue
1. Brain sections
2. 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 fromall foursegments
of the spinal cord: cervical, thoracic, lumbar,
and sacral, C1-L6 respectively.
Week 2
Week 3
Stain tissue
1. Primary antibody
2. Secondary antibody
3. Biotynyl tyramide
amplification
4. DAB blocking
 Sections, spaced 240 µmapart, will be stained
using an antibody to td-Tomato.
 Tissue will first be tagged using a primary
antibody.
 Between 1-3 daysof incubation are needed prior
to the addition of the secondary antibody.
Week 4  Processed tissue will be washed first with TBS
and prepared foraddition 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 sections
1. Microscope slide
mounting
2. 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
projections
1. Reassemble sections
2. 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

4. 4
ChristianJaquesHissom
Tuszynski lab,Center forNeuronal Repair
Chissom@ucsd.edu (805)-637-5653
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.