My research project involves the AP-3 (adaptor protein) complex, which plays a key role in membrane trafficking within cells. Vesicles (small, membrane bound structures) mediate the transport of proteins and lipids among cellular organelles. These vesicles are created in various ways by proteins throughout the cell, with some including a protein "coat" around the vesicle as it travels to its destination. AP-3 is a coat protein complex that mediates vesicular transport from the trans-Golgi network to the lysosome. We have utilized a gene reporter system called GNSI to analyze AP-3 function in different genetic backgrounds of Saccharomyces cerevisiae (baker’s yeast). One output of the reporter system is a colorimetric assay that measures the intensity of colored halos around yeast colonies grown on an agar plate. We found that targeted truncations of proteins of the AP-3 subunits Apl6 and Apl5 at the C-terminus of AP-3 (specifically the “ear domains”) resulted in increasing defects in AP-3 trafficking ability. However, it is unknown whether the defects are in membrane recruitment to the trans-Golgi network, or whether recruitment occurs but vesicle budding defects arise. Therefore, the current aim of the project is to begin analysis of the truncation defects in AP-3 by fusing Apl5 trunctions with a C-terminal mNeonGreen fluorescent protein and use live cell fluorescence microscopy to further examine AP-3 localization These basic studies will further our understanding of membrane trafficking and may provide insight into diseases linked to AP-3 function, including HIV-1 particle assembly and human genetic disorders.

Spinal cord injuries (SCI), unlike other injuries, often exhibit limited recovery. Patients with SCI often face major detriments to quality of life and health due to impaired motor control, sensation, and homeostatic regulation. In SCI patient surveys, regaining hand function is consistently among the highest priorities. We use a rat model of cervical SCI to develop therapies that restore motor function for reaching and grasping. We have shown that rats that receive targeted, activity-dependent spinal stimulation (TADSS) at a single spinal site for forelimb reaching exhibited enhanced recovery compared to physical retraining or open-loop electrical stimulation. Spinal stimulation is delivered to the injured forelimb of the rat when muscular activity, during reaching and grasping, is displayed. We used the single pellet grasping (SPG) task to assess forelimb function. Our hypothesis was that modifying the TADSS protocol to include stimulation of multiple motor pathways (MTADSS) for reaching and grasping will produce greater recovery than single site TADSS. In the current project, male and female Long-Evans rats were injured with a unilateral C4-C5 spinal hemi-contusion which primarily impairs the dominant forelimb. Four weeks after injury, the animals were implanted with spinal stimulation wires and wires for recording the muscle activity of the impaired forelimb. MTADSS and unstimulated control rats, underwent fourteen weeks of daily SPG, lever pull, which measures pull strength, and two other functional assessments, which will be reported elsewhere. MTADSS rats show recovery in SPG and a principal component analysis of lever pull showed that mean peak force (MPF), explains 84% of the variance between control rats and therapy rats at week 7 of therapy. The increased MPF among MTADSS rats suggests increased muscle recovery due to therapy. Taken together, MTADSS therapy seems to promote recovery in multiple measures of forelimb function compared to physical retraining alone.

More than half of spinal cord injuries (SCI) affect the cervical cord, which can have devastating life-long impairments to hand and arm function. We use a rodent model of cervical spinal contusion to develop activity dependent electrical stimulation therapy for regaining fine motor function after SCI. The Ohio State Injury Device uses an electromagnetic impounder to compress the spinal cord with a 2.5 mm probe and control variables that can affect the severity of the injury, most importantly displacement (i.e. how much the cord is compressed during injury). However, here we show that injury severity varies more than controlling for displacement can account for. We have also investigated the effect of surgeon on injury severity, which appears to have a small influence on injury severity. Upon further analysis, we have found that given similar probe displacements, male rats exhibit greater functional deficits after injury than female rats. Physiological differences such as size and hormone levels vary between males and females and may impact severity of motor function deficits. We hypothesize estrogen levels may influence injury severity. Previous studies have shown administering estrogen to a rat after SCI is neuroprotective and to decrease apoptotic activity in the spinal cord. In our ongoing studies, we will monitor the stage of the estrous cycle (a proxy for measuring estrogen and other hormones) in female rats prior to injury. Investigating the effects of estrogen on SCI helps us develop and understand injury models and motor function after SCI. A more sophisticated injury model may help us develop more effective treatments for SCI. Ultimately, results may help bring novel therapies closer to clinical use.

Spinal cord injury (SCI) is a debilitating condition that impairs motor function and overall quality of life. We have previously shown that Targeted Activity-Dependent Spinal Stimulation (TADSS) improves motor function in a rodent cervical SCI model. The hypothesized mechanism underlying TADSS is Spike-Timing Dependent Plasticity (STDP). During STDP, the strength of the synapse, or connection, between two neurons depends on the spiking behavior of a presynaptic neuron (A) relative to the postsynaptic neuron (B) within an optimal delay window. A synapse strengthens if A spikes less than 50 ms before B. Conversely, a synapse weakens if A fires less than 50 ms after B. It is currently unclear if TADSS strengthens corticospinal tract (CST) input into cervical spinal cord. In this project, we investigated the efficacy of TADSS therapy in inducing STDP in the rodent CST. TADSS therapy involved behavioral retraining of injured animals, with treated animals receiving concurrent spinal stimulation and control animals receiving no stimulation. In separate weekly sessions, the synaptic strength between motor cortex and spinal cord was assessed by measuring spinal cord evoked potentials (EPs) during test electrical stimulation. Test electrical stimulation involved current application to the forelimb region of motor cortex and recording of the EP response in cervical spinal cord caudal to the site of injury. After 3 weeks of TADSS, we observed larger EPs in TADSS animals and smaller EPs in injured control animals. In the weeks that followed, TADSS animals exhibited improved motor function while control animals exhibited declined motor function. Our results indicate increased connectivity between the motor cortex and spinal cord which precedes behavioral improvement -- this possibly suggests that strengthening the synaptic connectivity of the descending CST input to spinal cord is incorporated in the mechanism of TADSS-induced motor recovery.

Spinal cord injury (SCI) affects the lives of over 294,000 individuals in the United States alone. Therefore, there is an urgency for development of therapies for SCI. We are exploring the role of environmental enrichment in promoting motor recovery from chronic cervical SCI that produces partial to complete forelimb paralysis in adult rats. Novelty, a major component of our environmental enrichment, has been associated with memory consolidation which could be related to the release of plasticity-related products (PRPs). PRPs are a key component of lasting plasticity changes in vitro, which could prove to be vital to motor learning after spinal cord injury. Throughout a 6-week therapy period during which the rats are exposed to environmental enrichment, motor function of the impaired forelimb is assessed using behavioral scores on a reach-and-grasp pellet-retrieval task. Our project will utilize environmental enrichment to enhance the effectiveness of our physical training paradigm. Environmental enrichment will include access to toys that provide opportunities for physical exercise, socialization, and social learning. The toys will be changed each week to promote novelty. We predict that environmental enrichment will have an additive effect in promoting recovery of the impaired forelimb when combined with physical therapy. We hope these results will help inform how neural plasticity can be deployed for design of effective therapies for promoting motor recovery after chronic SCI.

Primates shift their line of sight using two types of eye movement. The first type, called saccades, is used to quickly bring an object of interest to the fovea. We use saccades to move our line of sight to the next words when we read. The second type is called smooth pursuit (SP). This movement smoothly tracks a moving object to keep it on the fovea. In real life, saccades often must be coordinated with other saccades or other types of eye movements that intervene between the programming and execution of a saccade. Such interruptions dissociate the vector of the saccade to be executed from its retinotopic target vector. The brain must update the vector of the upcoming saccade by combining the retinotopic target vector with information about the intervening movement. Many studies confirm that when the intervening movement is a saccade, the saccadic system compensated for the intervening movement so that the upcoming saccade reaches the target accurately. In my project, we used SP as the intervening movement. We presented a laser target spot step-ramp stimulus (30-60°/s) for 125ms after a fixation period. At the end of the ramp, a target was flashed for 25ms at a distance of 5° or 10° from the gaze location. The target was turned OFF for 600ms after that. In the dark, the monkey responded by making a SP movement and followed by a saccade. We expected that the saccadic system would compensate for the displacement of the eyes caused by the SP to arrive at the flashed target location, but the saccades missed the target location by the amount of the SP displacements. We conclude that the saccadic system fails to update the spatial relation between the visual target and the eyes when a smooth pursuit movement distorts it.

Motor injuries, such as stroke and spinal cord injury, are some of the leading causes of long-term disability and death worldwide. Patients are often left behind with debilitating consequences, such as paralysis or severe motor impairments, creating an urgent need for new therapies to be developed. A treatment option that is gaining momentum is the use of electrical stimulation to strengthen neural pathways for improving motor function of affected individuals. Towards this goal, I have investigated a novel electrical-stimulation paradigm called movement-triggered theta-burst stimulation (MT-TBS) to modulate neuronal connectivity in the motor cortex of macaque monkeys. To implement MT-TBS, a monkey is trained to perform a target-tracking task that activates wrist muscles involved in flexion and extension. While the monkey performs the task, sites in the motor cortex associated with wrist flexion or extension simultaneously receive theta burst stimulation; hence the movement triggers stimulation of cortical sites in this closed-loop system. After receiving MT-TBS, monkeys continue to perform the same target-tracking task on the days following conditioning. Early results show that 50% larger gains in cortical connectivity can be achieved on the same day of conditioning with open-loop TBS. This may indicate that MT-TBS may be an even better method for strengthening connections in the motor cortex. For my research project, I investigated the effects of MT-TBS on changes in the strength of neuronal connections in the motor cortex when coupled with repetition of the conditioned movement in the days following stimulation. In a series of experiments, MT-TBS was delivered during flexion, extension and with the muscle at rest. If successful, pairing MT-TBS with physical rehabilitation will serve as an effective strategy for aiding individuals with motor impairments.

The NMS, a physiological structure where neurons stimulate muscles via release of acetylcholine, is specifically organized to ensure efficient transmission of neural information by minimizing the diffusion distance of acetylcholine from the presynaptic neuron to the postsynaptic muscle cell. This is accomplished through the precise creation of ‘active zones’, which are neural cytosolic structures that anchor acetylcholine-containing vesicles directly across from acetylcholine-gated ion channels in the muscle cell. A failure to form active zones hampers synaptic transmission, causing muscle weakness disorders. Prior research has elucidated that active zone formation is predicated on the binding of the L5_III extracellular loop of presynaptic Ca_v2.1 to the beta-2 chain of laminin (a synaptic protein). My project is to create an improved assay measuring Ca_v2.1/beta-2 laminin binding. The eventual aim is to use the improved assay to elucidate the amino acids residues of the L5_III extracellular loop critical for binding laminin. To implement this assay, I created a beta-2 laminin construct (tagged with a red fluorescent protein) and built-up stocks of HEK 293 cells that can be transfected with DNA encoding Ca_v2.1 (tagged with the green fluorescent protein EGFP). Next, I cultured transfected cells and incubated them with 1 micrometer beads coated with the laminin protein construct. Using fluorescence microscopy, I then confirmed that beta-2 laminin does indeed bind to the Ca_v2.1 expressing cells by observing the co-localization of red and green fluorescence. Additionally, I have built up the necessary reagents to perform this binding assay with cells transfected with different Ca_v2.1 DNA constructs containing single amino acid mutations in the L5_III loop. If binding is no longer observed, the mutated amino acid(s) in the L5_III loop will be identified as being responsible for binding laminin. In conclusion, this project has paved the way for a greater understanding of the critical Ca_v2.1/beta-2 laminin interaction.