Project Proposal. Dentate-To-Thalamus Christian Jaques Hissom 4

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 motor task. 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 better understand behavioral anomalies in these animals.Mentor and department: Dr. James Conner, Ph.D., Dr. Mark Tuszynski, M.D., Ph.D. UCSD Center for Neuronal Repair

Proposal: Brief project description: 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 utilized and strengthened during the forelimb reach. How will I accomplish this study: Test subjects 18 rats will be trained and tested for accuracy and performance of skilled forelimb reach and cat-walk gait analysis. Surgery First surgery Insert viral vector (NeuRet) in thalamus to retrogradely infect to afferent projecting cerebellar dentate neurons with the human form of the IL2Ra receptor. Second surgery Inject immunotoxin specifically targeting the human IL2Ra receptor into dentate nuclei of cerebellum Clarity Behavior and training Post fist surgery- 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 skilled forelimb 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 successfully perform the 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 information is saved and later compared to post second surgery results. Post second surgery -Reassessment Rats will be reassessed in skilled forelimb reaching task a week after the second surgery. Once again, successful and unsuccessful attempts will be recorded and then compared to pre ablation scores. The rats gait and kinematics will be reassessed by doing a follow up cat-walk test. New results will be compared with pre-ablation scores. Histology Perfusion Rats will be perfused with formaldehyde and the tissue will be preserved for histology. 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 fluorescent protein. Clarity Sample tissue will be processed using the clarity protocol and wet-lab procedure the clarify tissue and prepare sample for imaging. Imaging Immunohitochemistry samples will be processed with lab microscope to assess specificity of target ablation. Clarity samples will be processed through UCSD core microscopes to assess intricacy of connection and observe physical changes caused by learning of skilled forelimb task. Motivation for research: 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 individual tract of the internal circuits.

Timeline -Week by week schedule

WeekObjectiveTasks Hours/ week

Week- 0 -Order rat subjects -Coordinate to shadow and learn clarity procedure. -Order 18 rats from Harland laboratory -Rats will take one week to arrive

15-20hrs per week

Week-1-Rats arrive - facilitate rats in Va Vivarian. - Establish feeding hours and training schedule.

Week-2-Surgery- inject Virus - Perform 2-3 surgeries per day - Each surgery will result in the injection of neuret viral vector into Va/Vl of Thalamus

Week-3Behavior-Training Forelimb reach-Training Cat-walk - Acclimate rats to training environment -Perform forelimb behavior training once a day for 2 weeks -Asses gait with Cat-walk, all rats on one day

Week-4Training Forelimb reach -Continue training

Week-5Surgery- Inject toxin - Anesthetize and surgically inject immunotoxin into dentate nuclei.

Week-6Reevaluate-Forelimb reach - Cat walk - Perform skilled forelimb reach each day for two weeks -Reevaluate Cat walk

Week-7Reevaluate -Forelimb reach -Catwalk - Continue forelimb reach- Assess Cat walk data and construct visual representation of data.

Week-8Histology-Immunohistochemistry -Clarity -Cut 40-micron brain slices and collect tissue.-Begin immunohistochemistry protocol -Begin Clarity protocol

Week-9Histology -Mounting and tissue processing -Clarity histology

- Continue immunohistochemistry -Continue clarity

Week-10-Clarity imaging - Weekend imaging

Budget worksheet Rat subjects, Immunotoxin, GFP

Items Quantity Supplier Cost

Test subjectsRats -18 rats-$55/rat USD

-Harland $990 USD

San Diego VA Medical Center Vivarion -$00.50/Rat/Day x 10 weeks -San Diego VA Medical Center $630 USD

Neuret vector and immunotoxin -25 micrograms

-Advanced Targeting Systems -Sigma-ALDRICH-VWR$210.00 USD

$204.50 USD $299.00 USD

Green fluorescent protein (GFP) antibody -100 micrograms

-Life Technologies $255.00 USD

Miscellaneous Immunohistochemistry And surgical supplies

$200.00 USD

Total

$2285

Clarity

Item Quantity Supplier Cost

Tissue Histology and clearing -3 UCSD CORE-Clarity $ 510.00 USD

Imaging Training UCSD CORE- Imaging $ 220.00 USD

Imaging processing -20hrsUCSD Core Imaging $ 400.00 USD

Total $1130.00 USD

Project total

Rat Subjects, Immunotoxin, GFP$2258 USD

Clarity tissue processing $1130.00 USD

Total $3388.00 USD

Budget justification Budget includes separate expenses for ablation procedure and clarity imaging 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 is covered prior to the imaging and the imaging can be resumed the preceding quarter. Rats and GFP antibody can only be purchased through the suppliers provided. This is because these suppliers work closely with the lab. Clarity protocol will be performed using the instruments and experimental tools available through the UCSD core. Dissemination

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 attempt to present the poster in as many future conferences as possible.