The proposed project aims to investigate the functional significance of the cerebellar dentate-to-thalamus pathway in learning and performing skilled motor functions. The researcher will conduct targeted ablations of this pathway in rats and assess the impact on motor performance and gait using behavioral assays, immunohistochemistry, and clarity tissue imaging. Comparison of control and ablated rats will provide insight into the role of this pathway in motor memory storage and the neural circuits strengthened through learning a forelimb reaching task. If successful, the results will be written into a research paper and presented as a conference poster to disseminate the findings.

1. 1ProjectProposal.Dentate-To-Thalamus–ChristianJaquesHissom
Investigating the functional significance of cerebellar dentate-to-thalamus pathway
in learning and performance of skilled motor function
Brief summary:
 The cerebellum plays an important role in mediating both coordination and storage of learned
motor sequences. This stored information, initiated by the frontal cortex, is retrieved by way of
the dentate-thalamic connection. The dentate nucleus rest within the cerebellum and receives
input from the intermediate lobe of the cerebellum. The cerebellum projects stored motor
sequence to the thalamus by way of the dentate-thalamic tract. Interaction and disinhibition of
globus palidus neurons by striatal input completes the initiation of the learned motor command,
which manifest performance of distal forelimb reach. In other words, initiation, coordination,
and performance of a motor command requires cross talk from a variety of nuclei, or neuronal
clusters, within the brain. This cross talk strengthens and forms circuit as the rat learns the
complex motortask.
It has been postulated that traces of the experience and the memory of the learned motor task,
in particular, reside within the intermediate lobe of the cerebellum. By using a novel cell-
ablation method, one can specifically infect the target tract with a foreign cell surface receptor
that will later bind a specific immunotoxin resulting in a selective ablation of neurons giving rise
to the cerebellar dentate-to-thalamus pathway. Pre and post ablation behavioral assays will
objectively determine changes in motor performance. Rats will be trained in a skilled forelimb
reach task as well as the cat-walk to assess kinematics associated with gait. In the past,
thalamocortical and corticospinal ablations resulted with rats demonstrating significant deficits
in performance on both of behavioral tasks. The data collected from this experiment will be
compared to performance of rats after thalamocortical and corticospinal ablation in order to
betterunderstandbehavioral anomaliesinthese animals.
Mentor and department:
 Dr. JamesConner,Ph.D.,Dr.Mark Tuszynski,M.D.,Ph.D.
 UCSD CenterforNeuronal Repair

2. 2ProjectProposal.Dentate-To-Thalamus–ChristianJaquesHissom
Proposal:
 Brief project description:
o The aim of this project is to perform targeted ablations of the cerebellar dentate-to-
thalamus pathway and assess the functional impact on skilled motor performance and
gait. Post ablation behavioral assays, immunohistochemistry, and clarity protocol will be
used to assay dentate-thalamic tract function and relevance to motor memory engram.
A comparison between control and ablated rats, by way of clarity protocol, will produce
a three dimensional representation of the connection and reveal the intricacy of circuits
utilizedandstrengthenedduringthe forelimbreach.
 How will I accomplish thisstudy:
o Test subjects
 18 rats will be trained and tested for accuracy and performance of skilled
forelimbreachandcat-walkgaitanalysis.
o Surgery
 Firstsurgery
 Insert viral vector (NeuRet) in thalamus to retrogradely infect to
afferent projecting cerebellar dentate neurons with the human form of
the IL2Ra receptor.
 Secondsurgery
 Inject immunotoxin specifically targeting the human IL2Ra receptor into
dentate nuclei of cerebellum Clarity
o Behavior and training
 Postfistsurgery- training
 Rats will be trained in skilled forelimb reach task. Each rat is first
acclimated to the testing environment. Rats are then trained to perform
skilledforelimb task, which requires extension of distal forelimb through
Plexiglas opening to retrieve a sugar pellet. Successful reaches are
Grabbing and successfully retrieving the sugar pellet categorizes a
successful reach. Incomplete reaches and inability to retrieve pellet
categorize unsuccessful attempts. After 10 days, all test subjects can
successfullyperformthe task.
 Rats will be first acclimated then trained to perform the cat walk. During
this test, each rat walks across a glass catwalk. Under the stage, a
camera records the animals gait and kinematic patterns. This
informationis savedand latercomparedtopostsecondsurgeryresults.
 Postsecondsurgery -Reassessment
 Rats will be reassessed in skilled forelimb reaching task a week after the
second surgery. Once again, successful and unsuccessful attempts will
be recordedand thencomparedtopre ablationscores.

3. 3ProjectProposal.Dentate-To-Thalamus–ChristianJaquesHissom
 The rat’s gait and kinematics will be reassessed by doing a follow up cat-
walktest.New resultswillbe comparedwithpre-ablationscores.
o Histology
 Perfusion
 Rats will be perfused with formaldehyde and the tissue will be
preservedforhistology.
 Immunohistochemistry
 Sample tissue from control and ablated rats will be cut at 40 microns
and the samples will be strained with anti-body staining and Green
fluorescentprotein.
 Clarity
 Sample tissue will be processed using the clarity protocol and wet-lab
procedure the clarifytissue andprepare sample forimaging.
o Imaging
 Immunohitochemistry samples will be processed with lab microscope to assess
specificityof targetablation.
 Clarity samples will be processed through UCSD core microscopes to assess
intricacy of connection and observe physical changes caused by learning of
skilledforelimbtask.
 Motivation for research:
o My motivation to peruse this research came from a previous experiment. Over summer,
I assisted with a thalamocortical ablation. I was introduced to the project and studied
articles related to thalamic ablations, specifically in the Ventral anterior, ventral lateral
nuclei of thalamus. This region of thalamus correlates most with motor performance
that receives input from Basal ganglia and Cerebellum. From my readings, I learned
more and more about the functions of the basal ganglia and cerebellum and I took
special interest in the intermediate lobe of the cerebellum. This region, which projects
to the dentate nucleus, has been shown to be a probable location for the storage of
motor command sequences. Furthermore, after collecting data from the thalamic tract
ablation, we were confronted with more questions than answers. It was difficult to
determine the source of the behavioral discrepancies observed after the ablation.
Further backtracking of both the Basal-thalamic and cerebellar-thalamic circuit will
provide beneficial information necessary to elucidate the specific function of each
individualtractof the internal circuits.

4. 4ProjectProposal.Dentate-To-Thalamus–ChristianJaquesHissom
Timeline -Week by week schedule
Week Objective Tasks Hours/week
Week- 0 -Orderrat subjects
-Coordinate toshadow and
learnclarityprocedure.
-Order18 rats fromHarland laboratory
-Ratswill take one weektoarrive
15-20hrs per
week
Week-1 -Ratsarrive - facilitate ratsinVaVivarian.
- Establishfeedinghoursandtraining
schedule.
Week-2 -Surgery- injectVirus - Perform2-3 surgeriesperday
- Each surgerywill resultinthe injectionof
neuretviral vectorintoVa/Vl of Thalamus
Week-3 Behavior
-TrainingForelimbreach
-TrainingCat-walk
- Acclimate ratsto trainingenvironment
-Performforelimbbehaviortrainingonce a
day for2 weeks
-AssesgaitwithCat-walk,all ratsonone day
Week-4 TrainingForelimbreach -Continue training
Week-5 Surgery- Injecttoxin - Anesthetize andsurgicallyinject
immunotoxinintodentate nuclei.
Week-6 Reevaluate
-Forelimbreach
- Cat walk
- Performskilledforelimbreacheachdayfor
twoweeks
-ReevaluateCatwalk
Week-7 Reevaluate
-Forelimbreach
-Catwalk
- Continue forelimbreach
- AssessCatwalkdata and constructvisual
representationof data.
Week-8 Histology
-Immunohistochemistry
-Clarity
-Cut40-micron brainslicesandcollecttissue.
-Beginimmunohistochemistryprotocol
-BeginClarityprotocol
Week-9 Histology
-Mountingandtissue
processing
-Clarityhistology
- Continue immunohistochemistry
-Continue clarity
Week-10 -Clarityimaging - Weekendimaging

5. 5ProjectProposal.Dentate-To-Thalamus–ChristianJaquesHissom
Budget worksheet
Rat subjects, Immunotoxin, GFP
Items Quantity Supplier Cost
Testsubjects
Rats
-18 rats
-$55/rat USD
-Harland $990 USD
San DiegoVA Medical CenterVivarion -$00.50/Rat/Day x
10 weeks
-SanDiegoVA Medical
Center
$630 USD
Neuretvectorandimmunotoxin -25 micrograms -AdvancedTargeting
Systems
-Sigma-ALDRICH
-VWR
$210.00 USD
$204.50 USD
$299.00 USD
Greenfluorescentprotein(GFP)
antibody
-100 micrograms -Life Technologies $255.00 USD
Miscellaneous Immunohistochemistry
Andsurgical supplies
$200.00 USD
Total $2285
Clarity
Item Quantity Supplier Cost
Tissue Histologyandclearing -3 UCSD CORE-Clarity $ 510.00 USD
ImagingTraining UCSD CORE- Imaging $ 220.00 USD
Imagingprocessing -20hrs UCSD Core Imaging $ 400.00 USD
Total $1130.00 USD
Project total
Rat Subjects,Immunotoxin,GFP $2258 USD
Claritytissue processing $1130.00 USD
Total $3388.00 USD
 Budget justification
o Budgetincludes separate expenses for ablation procedure and clarityimaging procedure.
I have placed this section separately due the possibility of budget constraints. While the
project would be most complete with the clarity imaging, a substantial amount of work
iscoveredpriorto the imagingandthe imagingcanbe resumedthe precedingquarter.
o Rats and GFP antibody can only be purchased through the suppliers provided. This is
because these suppliersworkcloselywiththe lab.
 Clarity protocol will be performed using the instruments and experimental tools available
throughthe UCSD core.Dissemination

6. 6ProjectProposal.Dentate-To-Thalamus–ChristianJaquesHissom
Dissemination
 I would like to compress my findings and produce a brief research paper with abstract, intro,
methods, discussion, and conclusion. Depending upon the nature of the findings from this study,
it is likely that follow-up studies will be added and the cumulative study will be submitted to a
peer reviewed scientific publication. I would also like to submit this paper for consideration of
publication in Journal of Undergraduate Research. After writing the paper, I would like to
reshape the text and present the information as a poster. I have presented at a poster
presentation once before and I enjoyed the experience more than I imagined. I was also
awarded first place in the ERN conference poster presentation. If this project is successful, I will
attemptto presentthe posterinasmany future conferencesaspossible.