Saccades are rapid eye movements that are essential for tasks like reading, and their accuracy is maintained through motor adaptation across life stages and in response to neural injuries of diseases. Our long-range goal is to identify the neural mechanisms of such saccade adaptation. While saccade adaptation has been intensively studied before, our understanding of the neurophysiological basis of this phenomenon is based largely on amplitude-decrease adaptation. However, the clinical relevance of amplitude-decrease adaptation is marginal at best, as overshooting saccades rarely occur in real life. Most erroneous saccades fall short of their target, necessitating an amplitude-increase adaptation. This is what we propose to investigate in this application. The challenge in studying amplitude-increase adaptation lies in the requirement of numerous saccades over extended periods to observe significant amplitude alterations. Our preliminary studies, conducted over daily sessions spanning more than a month, discovered a bimodal distribution of adapted saccades characterized by low and high gains, with gain defined as the ratio of saccade amplitude to the target step. Low-gain saccades surface initially, reaching peak velocity saturation leading to a gain increase saturation. Conversely, high-gain saccades emerged after extensive trials, characterized by their reduced velocities and prolonged durations, suggesting a novel adaptation mechanism through the fusion of consecutive saccade pairs. Our project aims to (1) characterize this newly found adaptation mechanism and (2) investigate the superior colliculus's (SC) role, which is pivotal in commanding saccade size, in generating both low and high-gain adapted saccades and the integration of saccade pairs. The overarching impact of this research lies in its potential to enhance our understanding of motor adaptation's role in recovering from motor deficits caused by neural damage. Understanding these changes may help refine the strategy of rehabilitation for patients with saccade dysmetrias, and perhaps motor hypometrias in general.

Proper chromosome segregation in mitosis relies on the correct attachment of kinetochores to the plus ends of microtubules. Kinetochores are protein complexes that assemble onto centromeres and bind microtubules. Microtubules are dynamic polymers of 𝛼- and 𝛽-tubulin subunits with an intrinsic structural polarity due to the repeated head-to-tail orientation of the heterodimer subunits in the lattice. This polarity results in a faster growing plus end and a slower growing minus end. Kinetochores are thought to initially bind to the microtubule lattice and then achieve plus end attachment by the action of plus end directed motor proteins or by the microtubule tip disassembling to the attachment point. While the plus end attachment is essential for mitotic fidelity, it remains unknown if the kinetochores themselves have an intrinsic polarity preference. Using total internal reflectance fluorescence microscopy, we have found that individual kinetochores assembled on centromeric DNA have a strong preference for binding the plus ends of stabilized microtubules in the absence of motor proteins and ATP or microtubule dynamics. Furthermore, using optical trapping we are able to measure the rupture forces of kinetochores on both ends of microtubules and have found that the observed preference for plus ends is matched by a greater binding strength at plus end tips. These results together give insight into how kinetochores could efficiently form plus end tip attachments and how they likely play a part in cell cycle regulation by using tension to sense a correct attachment. A better understanding of the specific mechanisms of kinetochore microtubule binding is valuable for understanding control of mitotic progression and could potentially inform more targeted anti-cancer therapies that focus specifically on dividing cells without impacting regular cell function.

Mitosis is the fundamental biological process that ensures equal separation of genetic material during cell division. Microtubules and their associated structures in the mitotic spindle execute this partitioning by carefully aligning and separating duplicated chromosomes. Despite intrinsically variable growth rates across microtubules and stochastic assembly and disassembly phases, chromosome-bound microtubules exhibit highly coordinated behavior that drive mitosis. The basis for this high degree of synchronization is currently unknown. Previously, we used a novel dual laser trap assay to show that microtubule pairs growing in vitro are coordinated by mechanical coupling (Leeds et al. 2023). A simple model incorporating both force-dependent pausing and growth speed heterogeneity explains the measured coordination of microtubule pairs. Our findings illustrate how microtubule growth may be synchronized during mitosis and provide a basis for modeling multiple microtubules in a bundle. In this project, we expand on the techniques we used with the dual optical laser setup and combine them with a cutting laser to induce disassembly in our microtubules. Studying the coordination of shortening microtubules encompasses a broader spectrum of microtubule dynamics and sheds light on other aspects of microtubule regulatory mechanisms. We can then extend our model to include the degree by which mechanical coupling can coordinate microtubules in disassembly in addition to growth. Bundles of multiple microtubules are in mixed states of shortening and growth while executing the coordinated motion necessary to drive mitosis, so understanding how mechanical coupling affects disassembling microtubules gives insight into the complete picture of the mechanisms behind their synchronous motion essential for life.

Cervical spinal cord injury (SCI) can severely limit motor functions of the arms and hands. With very few available and effective therapy options, SCI results in reduced independence and quality of life. Electrical stimulation of the spinal cord is a promising therapeutic method that improves motor function in individuals with a spinal cord injury beyond what can be achieved by spontaneous recovery. However, this improvement has primarily been measured by manually scoring motor-based tasks and it remains unclear how these functional improvements are represented in the control of muscle activity. In this study, we conducted differential, intramuscular electromyography (EMG) recordings in rats while they performed a forelimb reach and grasp task. These recordings targeted the tricep brachii, wrist extensors and digit flexors. Trials highlighted the initial, intermediate, and final stages of an eight-week therapeutic window during which the rats received targeted, activity-dependent spinal stimulation (TADSS). TADSS is based on the principles of spike-timing dependent plasticity to enhance the electrical activity of spared motor pathways. The stimulation protocol delivers intraspinal microstimulation in synchronization with functionally related motor movements. I hypothesized that rats with a unilateral, cervical contusion of the spinal cord receiving TADSS will exhibit changes in EMG patterns throughout therapy with improved muscle strength and coordination of activity across muscles. This would suggest that TADSS can improve the strength of signals traveling through the injured spinal cord to the muscles. Results also measure functional recovery using an objective method rather than subjective behavioral scoring addressing a methodology problem in the field. Future directions will be directed towards differentiating the precise mechanisms of motor improvement as an objective method of recovery analysis.

Chronic motor deficits are one of the most debilitating symptoms experienced by individuals with spinal cord injury (SCI). Use-dependent physical rehabilitation is the current standard of treatment after chronic injuries, but typically leads to marginal recovery of motor function, creating a need for interventions that target chronic motor damage. Our laboratory developed a neuromodulatory intervention that utilized targeted delivery of quipazine, a broad-spectrum serotonergic agonist, to improve motor function in a rat SCI model. Chronically-injured rats received physical rehabilitation combined with intraspinal delivery of quipazine. Our results show after a severe SCI, delivery of quipazine caudal to the lesion promoted substantial and long-lasting recovery, compared to physical rehabilitation alone, as assessed through changes in forelimb-motor performance. In the present study, we will use electrophysiology to assess changes in corticomuscular connectivity via motor-evoked potentials (MEPs) between motor-cortical areas in the brain and forelimb muscles. MEPs will be recorded in wrist flexors, wrist extensors, and triceps through chronically-implanted wires, and are elicited by electrical stimuli delivered to the motor cortex (via microwires in the rostral and caudal forelimb areas). An increase in MEP amplitude is indicative of strengthening corticomuscular connections. MEPs will be assessed before, during, and after a therapy period consisting of physical rehabilitation and delivery of quipazine. Additionally, MEPs will be recorded before the cervical contusion SCI to acquire pre-injury baselines. Weekly changes in MEP amplitudes, relative to pre-injury levels, will be compiled and compared to changes in motor performance. We hypothesize quipazine will increase motoneuron excitability and firing caudal to the lesion, which will strengthen corticomuscular connections between the motor cortex and forelimb muscles that were weakened by the cervical SCI. We expect increases in MEP amplitude will be correlated to behavioral improvements. Our experiments will establish a high-sensitivity metric for quantifying the electrophysiological mechanisms behind quipazine-driven motor repair.

Spinal cord injury (SCI) causes physical disability and chronic pain, but there can also be psychological issues like depression and/or anxiety. Clinically, the estimated rates of depression among the SCI population are from 11% to 37%, according to UW Medicine. In rodents, after SCI, both males and females demonstrated anxiety-like behavior, and female mice became more anxious while male rats became more hypersensitive to thermal stimuli. These findings highlight the complexity of the systematic changes after SCI, all of which may impact the quality of life and limit functional recovery. We have found a sex difference in the effectiveness of electrical stimulation in promoting functional recovery after cervical SCI. In our experiment, females show robust functional improvements with activity-dependent spinal stimulation. The current study aims to investigate the role of affective behaviors (depression and anxiety) and pain after SCI on functional recovery in male and female rats. Before injury, all rats will undergo baseline assessments to establish behavioral norms, which involve training and evaluations designed to measure motor ability, emotional state, and sensitivity to various stimulations. Three weeks after SCI, rats will be assessed with the same battery of tests and then start daily treatment of drugs that can modulate emotional states and relieve pain, including a mixed serotonin and norepinephrine reuptake inhibitor, duloxetine (or no drug control), for five weeks. Behavioral assays will be repeated at the end of the treatment period, and tissue will be collected for histological analysis. I will primarily be responsible for conducting and analyzing an assay of anxiety-like behaviors, the open field test, and the assay of depression-like behaviors, the sucrose splash test. We expect to understand better the relationship between affective responses and motor function post-SCI, and the potential therapeutic benefits of antidepressant treatments, and particularly identify sex differences that may limit recovery.

The heart is one of the most mechanically active organs in the body. In a mechanism known as the “Bainbridge Reflex”, the heart rate accelerates in response to the mechanical stretch induced by the increase in venous return. The cardiac pacemaker controls heart rate, and while stretch-activated channels have been identified in cardiac tissue, their molecular identity remains unknown. We hypothesize that PIEZO channels are the molecular determinant of the stretch-dependent heart rate acceleration responsible for the Bainbridge reflex. Using quantitative polymerase chain reaction (qPCR), we assessed the presence of Piezo1 and Piezo2 transcripts in the pacemaker, atrium, and ventricle of the mouse heart. Our findings revealed that both Piezo1 and Piezo2 are present in the three regions with significantly higher expression in the pacemaker and atria. Combining immunohystochemistry, tissue clearing, and super-resolution microscopy, we analyzed the distribution of Piezo1 and Piezo2 in mouse pacemaker explants. Our results show that Piezo2 is uniformly expressed in the pacemaker and surrounding atrial tissue, whereas Piezo1 exhibits higher expression levels outside the pacemaker. These results were further confirmed at the single-cell level, with immunostaining of Piezo1 and Piezo2 in isolated pacemaker cells (HCN4+) and transitional cells (HCN4-). We observed similar expression levels of Piezo2 in both cell types and increased Piezo1 expression in transitional cells. In addition, we observed distinct localization patterns for Piezo1 and Piezo2 at the subcellular level. Piezo1 predominantly localizes to the sarcolemma, while Piezo2 exhibits a striated distribution that colocalizes alternately with both the Z- and the M- line of the sarcomere. Given this pattern, half of the Piezo2 bands colocalize with the RyR. These results set the starting point to evaluate the functional role of PIEZO channels in the cardiac pacemaker.

Spasticity is an increase in muscle tone (hypertonus) and abnormal muscle stiffness that impedes functional activity. Oftentimes observed among individuals with chronic neurological conditions such as traumatic brain or spinal cord injury (SCI), spasticity develops as a result of damage to the central nervous system (CNS). This damage disrupts the balance of supraspinal inhibitory and excitatory inputs to the spinal cord, which can lead to the loss of inhibitory inputs and hyperexcitation of the spinal reflex arc. The aim of this project is to develop an electrical stimulation protocol that regulates imbalances of supraspinal input and the spinal reflex in order to potentially alleviate spasticity caused by traumatic neural injury in patients. The hyperexcitation associated with spasticity is measured using the Hoffman-reflex (H-reflex). Previous studies have revealed that electrical stimulation of the rat motor cortex can modulate long-term spinal excitability. In this study, behaving noninjured Long Evans rats are implanted with cortical implants to induce stimulation to the motor cortex, grounding electrodes to filter environmental noise, cuff electrodes to evoke the H-reflex, and EMG electrodes to record the H-reflex response. The H-reflex is assessed by stimulating a cuff electrode surrounding the median nerve and measuring the consequent activity of EMG electrodes that are implanted into the flexor, extensor, and tricep muscles before and after electrical stimulation of the motor cortex. Our preliminary results indicate that different frequencies of cortical stimulation can modulate the H-reflex, suggesting that our novel cortical stimulation protocol may reduce spasticity and promote restoration of motor function. In future studies, I plan to assess the efficacy of cortical stimulation for improving spinal excitability in spastic animals following chronic SCI.

Impairments in memory formation are commonly observed during aging, even in the absence of disease-related neuropathology. However, we currently lack a comprehensive animal model of normative aging. Monkeys have a keen ability to remember pictures they have seen before, and this memory can be quantified through the tracking of eye movements. Previous research from the Buffalo Lab has shown that monkeys, like humans, show distinct patterns of eye movements when they first view an image, compared to when they view it during a second presentation. To investigate whether these behaviors are impacted in normative aging, I analyzed data from 6 young Macaca mulatta (4 female, 2 male, aged 7.5 ± 2.1 years) and 3 aged (female, aged 21.3 ± 2.5 years). In each block of trials, monkeys were shown 12-16 complex full-screen images on a computer monitor for 5-7 seconds of cumulative looking-time. If the monkey looked away from the screen, the picture remained on screen, but this time was not counted towards the cumulative looking requirement. Each behavioral session contained 5 blocks of trials, for a total of 60-80 unique images, each shown twice per session. I quantified attention as the total time the picture was onscreen compared to the required cumulative looking time, creating a ratio (or percent overage) for both novel and repeat images. Thus, the more time the monkey spent looking away from the image, the larger the ratio, representing less attention to the image. The data suggest that aged monkeys exhibit less attention to repeated images compared to novel images and less attention to repeated images than young monkeys. These differences in viewing behavior for repeated images may be indicative of age-related changes in memory processes, and this metric has the potential to better inform mechanisms of normative aging.

Spinal cord injury (SCI) causes significant sensorimotor deficits that negatively impact autonomy and quality-of-life. In a previous study, we determined that optogenetic spinal stimulation significantly enhanced forelimb recovery, axonal growth, and angiogenesis compared to sham controls. However, we have yet to determine the synaptic changes associated with optogenetic stimulation after SCI. The current project addresses this important detail by quantifying the synaptic changes that occur with and without optogenetic spinal stimulation in rats with cervical SCI.To investigate this, rats received a moderate hemicontusion of the 4th cervical segment (C4) and a spinal injection of an optogenetic viral vector (AAV2-hSyn-ChR2-YFP) to express light-sensitive proteins in the ipsilateral sixth segment (C6). Four weeks later, rats received a second surgery to receive a blue uLED implanted over ipsilateral C6 for optogenetic or sham stimulation. Rats were trained and scored regularly on a variety of forelimb behavioral tasks throughout the course of the study, while the rats in the stimulated group received stimulation 1x/week for 6 weeks beginning on the 6th week post-injury. After perfusion, the cervical spinal cord was sectioned and underwent immunohistochemistry (IHC) staining to examine synaptic density around motoneurons caudal to the lesion site where stimulation or sham stimulation occurred. Synaptic quantification has been completed using FIJI software. Our initial results reveal increased synaptic density around the motoneurons of rats that received optogenetic spinal stimulation, suggesting an increase in synaptic plasticity and connectivity. This indicates that stimulation not only enhances axonal growth but also supports the formation of new connections with downstream neurons partially disconnected by the injury. This study provides insight into the circuitry-related changes involved in SCI recovery. Identifying specific mechanisms of how optogenetic stimulation improves recovery can guide the development of more effective stimulation paradigms and treatment strategies in order to optimize functional recovery for people with spinal cord injury

As we receive spatial and temporal information, our brain develops sequential patterns to store events, giving us the ability to learn and store relationships. For learning and memory, these rapidly-encoded sequences are reactivated as “replay” sequences after experiencing the original trajectory, as often observed in the hippocampus. This brings up the question: what biological mechanisms enable us to build, encode, and trigger these relationships and replays? The goal of this project is to model sequential replay in spiking neural networks to explore and understand various biological mechanisms that produce the acquisition of such sequences. We are using NEST Simulator, a spiking neural network simulator software, to model large-scale neural networks. Then, we explore how changing dynamics such as non-random structure of the network and interactions between excitatory and inhibitory cells can contribute to sequence generation, as well as the salience and speed of such sequences. We have observed the significance of interplay between particular parameters, such as the widths of spatial Gaussian distributions for neuron connection strengths, by analyzing generated spiking raster plots. Recent work has also suggested an important role of long-term potentiation of intrinsic excitability in sequential replays, which we are integrating with the aforementioned dynamics by building a unique synapse model within the simulation software. This is a novel method to introduce excitability in a network, which is important to determine how changing excitability through potentiation, rather than plasticity, facilitates network formation and propagation. This research is significant because it highlights the components of neural networks that could be crucial to quickly generating and maintaining sequences for learning and memory, therefore helping us understand the brain’s mechanisms for storing spatiotemporal relationships.

BK channels are potassium channels activated in response to depolarization and elevated intracellular calcium ion levels. It has been observed that BK channels form clusters in cells, but the mechanism for clustering has not been characterized. This project attempts to discover important components that lead to BK channel clustering using super-resolution microscopy, proximity ligation assay, and Fluorescence Recovery After Photobleaching (FRAP) experiments. One possible mechanism relates to denser regions in the plasma membrane of PtdIns(4,5)P₂ as a possible lipid raft, hypothesized to localize proteins. We used tsA-201 cells to express BK channels (α subunit). In FRAP experiments, BK channels were tagged with a green fluorescent protein (GFP). To modify the levels of PtdIns(4,5)P₂, we co-expressed PIP5Kγ, the enzyme that catalyzes the synthesis of PtdIns4P to PtdIns(4,5)P₂. Expression of PIP5Kγ is known to increase PtdIns(4,5)P₂ levels by 30%. In our analysis, we assumed that large, bright fluorescent dots in live cells correspond to BK clusters. We found that co-expression of PIP5Kγ with BK decreases cluster size by 43% in super-resolution experiments and increases the number of puncta (BK clusters) by 41% in PLA experiments. FRAP experiments on a PIP2 biosensor, PH-PLCδ1-GFP, showed reduced fluorescence recovery speed when PIP5Kγ was co-expressed. Future FRAP experiments observing BK channels will allow us to determine if membrane components, such as PtdIns(4,5)P₂, influence the integrity and mobility of BK clusters and if the addition of these lipids is sufficient to induce additional cluster formation.