Hearing loss affects approximately 37.5 million adults in the United States and is associated with significant comorbidities, including depression, anxiety, and social isolation. Among the various etiological factors, aminoglycoside antibiotics are an important contributor to irreversible hearing loss due to their ototoxic effects on inner ear hair cells. Upon entry neomycin accumulates in the cytoplasm and lysosomes, and induces an acute hair cell death. G418, on the other hand, accumulates in lysosomes before triggering delayed death. This difference suggests a previously underexplored role of lysosomal signaling in hair cell survival. Due to the difficulties to access the mammalian inner ear, we are using the larval zebrafish lateral line to study live hair cells. My study investigates the role of lysosomal calcium release in mediating protection against aminoglycoside-induced hair cell damage. Specifically, we focus on Two Pore Channel 2 (TPC2), an ion channel located on the lysosomal membrane, which allows for calcium release upon activation by the agonist TPC2-A1-N. Using dose response curves, we examined the effect of TPC2-mediated calcium release on hair cell survival following G418 exposure. Our findings indicate that activation of TPC2 is able to protect against G418, but not neomycin. Moreover, protection is time sensitive, since activating TPC2 before G418 accumulation confers protection to hair cells, whereas co- and post-exposure activation does not yield a protective effect. These results suggest that lysosomal calcium release plays a critical role during aminoglycoside-triggered delayed hair cell death. This study provides novel insights into lysosomal calcium signaling as a potential mechanism for mitigating aminoglycoside ototoxicity and highlights TPC2 as a promising therapeutic target for hearing loss prevention.

All mammals experience a slowdown of cardiac pacemaker rate with aging. The main mechanisms to explain that phenomenon are related to alterations in the ionic currents that underlie the diastolic depolarization phase of the action potential. We have previously reported that pacemaker cells from old mice have reduced L-type calcium currents. We further explore the mechanism underlying that reduction, testing cell hypertrophy and alteration in the scaffolding of L-type calcium channels as potential mechanisms. To test for cell hypertrophy, we combined immunostaining and high-resolution imaging to map the HCN4-positive pacemaker region of isolated upper heart explants from young and old mice. We compared cell length, width, and area between young and old cells. We also determined these morphological parameters in HCN4-positive enzymatically dissociated pacemaker cells. We found no significant difference in cell dimensions or area between ages, ruling out hypertrophy as a potential mechanism. We used mass spectrometry to identify expression changes in scaffolding proteins essential for calcium channel organization at the plasma membrane. Through this approach, we identified a large reduction of caveolin 3 as a possible mechanism. Caveolin is a protein essential to forming signaling microdomains between calcium channels and other proteins. Using western blotting, we confirmed a 50% reduction of caveolin 3 in isolated pacemaker tissues from old animals. Using proximity ligation assay and super-resolution microscopy, we showed altered recruitment of L-type calcium channels into caveolae. Our findings suggest that the age-associated decrease of L-type calcium current is caused by a reduced insertion of these channels in caveolae.

The heart's primary function is to pump blood to supply oxygen and nutrients to the body. The biomechanical principles of the heart are determined by specializations at the organ, tissue, cellular, and molecular levels. Little is known about how these specializations have adapted to sustain high heart rates in animals with extreme biology, as is the case of the hummingbird, whose heart rate above 1000 bpm makes it the endotherm with the highest heart rate observed in nature. We hypothesize that the hummingbird heart has evolved several adaptations at all the abovementioned levels to i) generate fast firing rates, ii) optimize electrical-contraction coupling, and iii) sustain fast contraction-relaxation cycles. Using different histological and imaging approaches, we have started to characterize the architecture of the hummingbird’s heart for the first time in a research lab. To describe the overall dimensions and structure of the hummingbird heart, we generated CT scans and 3D reconstructions of iodine-labeled Calypte anna hummingbird hearts. To characterize the organization of the tissue, we present data using hematoxylin-eosin and lectin stainings in fixed paraffin-embedded slices of the hummingbird heart. Our preliminary results showed that hummingbird ventricles have a cell density of 110 cells per 5000 µm2, around 7-fold larger than mouse ventricles. Ventricular cells in the hummingbird are 8-fold smaller with a cross-sectional area of 41 ± 4 µm2. Hummingbird hearts also have a higher capillary density with 18.0 ± 0.6 capillaries per 2500 µm2. Our results provide a foundation for structural and functional characterization of the hummingbird heart at an organ, tissue, and cellular level while opening avenues for further investigation of extreme cardiac physiology.

Dynamics of activity across the cerebral cortex at the mesoscopic scale – coordinated fluctuations of local populations of neurons — are essential to perception and cognition and relevant to computations like sensorimotor integration and goal-directed task engagement. However, understanding direct causal links between population dynamics and behavior requires the ability to manipulate mesoscale activity and observe the effect of manipulation across multiple brain regions simultaneously. Here, we develop a novel system enabling simultaneous recording and manipulation of activity across the dorsal cortex of awake mice, compatible with large-scale electrophysiology from any region across the brain. Transgenic mice expressing the GCaMP calcium sensor are injected systemically with an adeno-associated virus driving expression of the ChrimsonR excitatory opsin. This strategy drives expression of the blue-excited calcium indicator, GCaMP, in excitatory neurons and red-excited Chrimson opsin in inhibitory neurons. The light channels of the imaging and the opsin do not interfere. We demonstrate widefield single-photon calcium imaging and simultaneous galvo-targeted laser stimulation over the entire dorsal cortical surface and find that the spatial and temporal resolution of the stimulus is suitable for targeting many specific cortical regions in short periods of time. The calcium indicator responded to the laser within 30 ms, and the activity returned to baseline within 100 ms after laser offset. The area of effect was as small as 3 mm² for the lowest laser power or as large as 10 mm² for the largest laser power. Moreover, the preparation is stable over many months and is thus well-suited for long-term behavioral experiments. The ability to stimulate and measure anywhere on the dorsal cortical surface of the brain will allow us to design computational models describing how causal manipulation impacts neural dynamics, especially in the context of designing closed-loop systems to control neural activity and behavior. 

Sensory signal processes (specifically how visual systems perform) function under the limits imposed by physics. One such physical limit comes in the detection of light. Light is divided into discrete amounts of energy called photons. Because of this division, light is inherently variable and operates under such variance sets on the retina. So far, the only thing studied in low light is if our system can detect the existence of a flash or dim light, but not deduction of movement and time information. Through measurements of the flicker fusion frequency, rod cells have been understood historically as having extremely poor temporal resolution. This suggested that the ability to detect moving objects– which relies on timing information– would be poor at low light levels. Through extracellular electrophysiological experiments conducted using a 512-channel multi-electrode array, I recorded the electrical activity of neural firing from a population of retinal ganglion cells in the primate retina to show that there is high temporal precision in star-light levels due to compensatory mechanisms in the retinal ganglion cells and adjunct circuits. Since we currently do not yet have any definitive understanding of the cellular-level population dynamics that explain the internal mechanisms and physiology in the retina that allow for this behavioral sensitivity and adaptation, developing this understanding of how a population of cells in the retina work together to detect and encode the motion of moving objects during a given time interval will be pertinent to furthering the field’s understanding of the internal mechanisms of the retina.

Cervical traumatic spinal cord injury (TCSCI) is a devastating condition that leads to tetraplegia, severely impairing essential life functions and independence. Individuals with cervical TCSCI struggle with hand function, reaching, eating, grasping, and writing, significantly reducing their quality of life. In the U.S., cervical SCI is the most common type of spinal injury, affecting over 300,000 individuals, with approximately 17,900 new cases annually. The long-term disability resulting from TCSCI often necessitates continuous medical care, rehabilitation, and assistive technologies to enhance functional recovery. Our preclinical study evaluates upper extremity dysfunction in rats following cervical TCSCI using behavioral assessments, specifically the Forelimb Reaching Task (FRT) and the Irvine, Beatties, and Bresnahan (IBB) test. These tests provide valuable insights into motor impairments and recovery over time. FRT assesses shoulder movement and fine motor control by placing the rat in a transparent box with side slits, allowing it to extend its forelimb to grasp a chocolate pellet. The grasping behavior is scored on a standardized scale. This test primarily evaluates digit precision and reaching ability. IBB provides a broader analysis of forelimb function, including both proximal and distal limb recovery. In this test, the rat is placed in a cylinder with food, and its grasping and eating behavior are recorded. Forelimb function is later evaluated based on elbow position, paw support, forepaw placement, and digit movements. By comparing these tests, we aim to determine their efficacy in assessing functional deficits and recovery post-SCI. This analysis is critical for refining behavioral assessments and guiding the development of new therapies to enhance motor recovery and improve the quality of life for individuals with cervical SCI.

Fentanyl is a synthetic opioid that has become the leading driver of the U.S. opioid epidemic, contributing to over 70,000 overdose deaths annually. Opioid use disorder (OUD) is characterized by cycles of dependence, withdrawal, and relapse, with most fatal overdoses occurring during relapse, yet existing treatments for OUD do not effectively prevent relapse. Understanding how fentanyl affects brain activity and behavior is critical for developing more effective therapies. I investigated how fentanyl exposure modulates locomotion and the neural activity in the nucleus accumbens (NAc) across abstinence, dependence, withdrawal, and relapse. I hypothesized that each stage would show distinct neural activation patterns and that fentanyl exposure would reduce exploration and locomotion, reflecting compulsive drug-seeking behavior. To test this, I implanted silicon probes in the NAc of mice to monitor neural activity while tracking movement and behavior with high-resolution video. Mice received increasing fentanyl doses over five days, followed by a withdrawal period and, finally, a relapse challenge dose. I analyzed their behavior using deep learning-based pose estimation for correlations with neural activity across different stages of fentanyl exposure. I expect neural recordings to show that fentanyl significantly alters NAc activity, with each phase displaying unique neural patterns. I also expect fentanyl-exposed mice to show reduced exploratory movement, consistent with behavioral inflexibility and compulsive drug-seeking tendencies characteristic of OUD. These findings could provide critical insights into how fentanyl disrupts brain function and behavior, helping to identify new targets for addiction treatment. This research lays the groundwork for future studies on relapse prevention, with the goal of improving OUD therapies and reducing overdose deaths.

The mechanisms guiding the sensory detection of pain and the subsequent sensitization of damaged tissue to mechanical and thermal stimuli are relatively well understood. However, mechanisms guiding the transformation of nociception into the negative feelings associated with pain remain largely unknown. This affective component, notably in chronic pain, translates into an intense emotional impact on patients and can contribute to the development of comorbid psychiatric disorders. The elderly population have a propensity to be socially isolated and face exacerbated effects of chronic pain. In 2021, an estimated 20.9% of U.S adults suffer from chronic pain with persons over 65 years of age having the greatest propensity of acquiring the disease. Due to this, clinical intervention models call for a more holistic approach to pain intervention that incorporates lifestyle and nutritional factors, extending beyond pharmacological treatments. One of these promising non-pharmacological interventions is positive social interaction, which has been shown to alleviate pain and suffering.  Several studies show that humans who maintain strong social bonds recover from injuries faster than people without them. However, it has not yet been evaluated the extent to which this phenomenon occurs in geriatric animals and its relative efficacy as a social intervention to alleviate chronic pain in injured mice. My project seeks to gauge whether social intervention can alleviate chronic pain symptoms in aged mice and to unveil the underlying mechanisms guiding these successful non-pharmacological treatments. I will achieve this through two aims: evaluation of social self-administration as an intervention for chronic pain, and transcriptomic analysis to identify gene expression changes as a result of social interaction. Future research will include miniscope endomicroscopy recordings to visualize cell activity within major brain regions, and comparison of cell ensemble activity between groups of mice will lead to the identification of structures encoding behavioral shifts caused by pain.

Integrating complex animal behavior with peripheral physiological recording is critical for revealing the neural basis of behavior. Traditional peripheral physiological recording methods constrain natural behavior due to cable tethers, and manually annotating behavior often introduces subjectivity. We have recently published two pipelines that independently overcome these confounds: (1) mechano-acoustic (MA) devices that provide wireless, minimally invasive peripheral recording based on finely-tuned accelerometers, and (2) a computer vision based machine learning package (Simple Behavioral Analysis, SimBA) for supervised behavioral classification from recorded videos. Here, we developed a comprehensive machine learning model to classify behavioral states using MA device accelerometer data, using SimBA to validate and extend model outcomes. We test this model by analyzing the effect of anesthesia and other consciousness-altering drugs on mice. Lastly, we extend this approach for closed-loop applications. This work contributes to the growing field of bio-signal processing, offers a data-driven approach to automated behavior classification, and provides the groundwork for answering many diverse questions in neuroscience and related fields.

A subset of voltage-gated potassium channels, Kv2s, are responsible for the majority of the perisomatic delayed rectifier current in pyramidal neurons of the neocortex. Mutations in these ion channels and their associated proteins cause developmental epilepsy, but the cellular mechanisms underlying this remain less clear. Previously, we have shown that the two members of the Kv2 family of voltage-gated potassium channel α-subunits, Kv2.1 and Kv2.2, are expressed differently depending upon the type of neuron in rodent primary sensory and motor neocortex. There are two major subclasses of layer 5 (L5) pyramidal neurons in the neocortex, extratelencephalic (ET) and intratelencephalic (IT) neurons, that are distinguished by their projection targets and laminar distribution. ET neurons, enriched in L5b of the neocortex, send projections to subcortical structures, whereas IT neurons, primarily located in L5a, project within the telencephalon. In rodents, ET neurons are enriched in Kv2.1, but not Kv2.2. Here, we tested whether these features extend to the association cortices of primates, particularly the prefrontal cortex and temporal cortex, which are essential for various higher-order cognitive functions, including recognition, attention, and planning. Using immunohistochemistry against Kv2.1 and Kv2.2, we showed that these subunits have distinct laminar distributions in the dorsolateral prefrontal cortex (dlPFC) and temporal cortex (TCx). Kv2.1 was predominantly expressed in L5b, whereas Kv2.2 was more concentrated in layer 2 (L2) and L5a. Using a tarantula toxin, Guanxitoxin (GxTx), to block the Kv2-mediated current, we found that, similar to what we observed previously in rodents, the role of Kv2 channels differs depending on the L5 neuron type. GxTx makes L5 ET neurons fire repetitive bursts, whereas GxTx makes L5 IT neurons less excitable. Together, these results support distinct roles for Kv2.1 and Kv2.2 in regulating excitability across ET and IT neurons in the association cortex of the macaque. 

Age-related macular degeneration arises from irreversible photoreceptor loss. Photoreceptors, rods and cones, are specialized cells in the retina that allow light and color detection. My project investigates the role of retinoic acid (RA) on cone and cone-opsin development to understand the timeline of cone specification and development. RA, an endogenously synthesized vitamin A derivative present in the retina during development, drives rod photoreceptor differentiation, but its effect on cone development is still unknown. To understand RA’s role in opsin development, I use a retinosphere (RS) model, an in vitro system to culture human fetal retina. More specifically, I used RS from 70 to 90 days old (D70-D90) and cultured the RS until D100, when the cone-opsin onset occurs. I then fixed, cryosectioned, and immunostained the two conditions for S-opsin, M/L opsin, and NR2E3 (rod marker) and investigated changes in the density of cone opsin-positive cells between the two conditions using confocal microscopy. My findings showed that the condition containing exogenous RA had a decreased density of opsin-positive cells. To confirm that the observed effect is due to RA, I mimic the experiment by instead using WIN18446, an RA inhibitor. I then determined if RA's effects are dose-dependent. My results showed that increasing the concentration of exogenous RA amplified my previous findings. The next step is to understand the timeline of cone specification and development by using RS of a younger age, before cone-opsin onset. These results will allow my mentors and me to use our knowledge about RA to determine if inhibiting endogenous RA synthesis in the retina will play a role in developing therapeutics involving cone regeneration to aid in cone-related macular diseases and injuries.

Correct segregation of chromosomes in cell division relies on kinetochores forming end-on, bioriented attachments to microtubule plus ends. In vivo, kinetochores are known to first bind to the lattice of the microtubule and then transit to the plus end either by tip disassembly or the action of plus end directed motor proteins. Force spectroscopy has recently revealed that kinetochores grip the microtubule lattice asymmetrically. Only ‘on-path’ kinetochores that are pulled toward the microtubule plus end form strong, load-bearing attachments, while minus end directed kinetochores weakly grip the lattice. The weak grip of minus end directed kinetochores limits tension across sister kinetochores and makes them susceptible to detachment by error correction machinery. We seek to investigate the molecular mechanism underlying the asymmetric grip of the kinetochore. We purified recombinant kinetochore subcomplexes and tested them individually for asymmetry. We show that the Ndc80 complex exhibits a similar asymmetry as the kinetochore, albeit weaker, while the Dam1 complex is ambivalent to microtubule polarity. Single molecule fluorescence microscopy shows that kinetochores pulled toward the minus end of microtubules are deformed relative to plus end directed kinetochores. We propose that the asymmetric grip strength of kinetochores arises from a network of interactions between polar-sensitive and polar-insensitive subcomplexes that is disrupted when the kinetochore is pulled toward the minus end of a microtubule. 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.

Artificial neural networks (ANNs) are now able to learn from data to recognize patterns, often equaling or exceeding human performance. If we can understand the learned internal representations of such networks, we stand to gain insights into the brain. By taking a visual neurophysiologist’s approach to studying internal representations in deep convolutional neural networks (CNNs) trained to solve challenging visual recognition tasks, I aim to further our understanding of the primate visual system. To do this, I have taken sets of visual stimuli used by neurophysiologists to study the encoding of shape, texture and color in the mid-level visual cortex of macaque monkeys and presented them to CNNs (e.g., ResNet and AlexNet).  I found that activations of units within CNNs show a higher level of invariance to changes in surface properties of simple shapes (e.g., shape selectivity remains consistent when filled shapes are replaced by their outlines) than do cortical neurons. I also found a correlation between this invariance and the sensitivity of units to shape vs. texture stimuli that holds up in several CNNs.  Specifically, units with lower invariance to surface properties tend to respond with a wider dynamic range to textures than to shapes.  If this holds in other classes of visual ANNs, it could establish a general principle for mid-level visual encoding in which the surface properties (texture and color) of an object are represented somewhat distinctly from the position and shape of the boundary of the object.  This is consistent with the observation in the primate visual cortex that some neurons specialize in texture encoding, while others specialize in shape encoding. Ultimately, a better understanding of how information is encoded and processed in the cerebral cortex can allow us to build devices that interface better with the brain and to someday address brain disorders.

The retina is a layer of neurons on the back of the eye that sense light and relay visual information to the brain. Our goal is to understand the role of epigenetic repression in retinal cell development by focusing on the polycomb complex, a complex of many proteins that repress gene expression through deposition of the H3K27me3 mark on histones. The goal of my project is to learn how the polycomb complex influences retinal development by altering specific aspects of the complex’s activity and observing how these alterations influence cell fate, using two complementary model systems: fetal-derived retinospheres and stem cell-derived retinal organoids. To perturb different aspects of the polycomb complex, I have treated retinospheres with Gskj4, a UTX inhibitor, and BRM014, a BAF inhibitor. During development, UTX is responsible for removing H3K27me3 so genes that are silenced can be expressed. When I added Gsjk4 to 135-day old retinospheres, I observed that cell proliferation decreased, and more cells expressed the marker OTX2, indicating an upregulation of either bipolar or photoreceptor cell differentiation. These data indicate that H3K27me3 removal is critical for proper specification of retinal cell types. BRM014 inhibits BAF, an ATP-dependent chromatin remodeler that has been shown to be recruited by UTX to remove nucleosomes and initiate transcription. When I added BRM014 to day 135 retinospheres, I also observed an increase in the expression of OTX2, similarly indicating an upregulation of either bipolar or photoreceptor cell differentiation. From these experiments, we conclude that removal of H3K27me3 is necessary for proper retinal cell specification and development. A better understanding of epigenetic regulation during retinal development will allow us to develop therapies to regenerate damaged retina lost in blinding diseases and restore sight to patients.

How the brain transforms sensory information to guide action and choices remains largely unknown. Although the brain regions and systems involved in decision-making are studied extensively in primates, understanding the details of the neuronal cell types and circuits that perform the computations related to decision-making requires the use of an animal model that is amenable to neuronal cell type-specific and circuit-specific manipulation. The mouse (Mus musculus) has become a model of choice for such experiments due to the explosion of new genetic and molecular tools allowing for such experiments. However, the behavioral sophistication of the mouse model is very different from that of the primate, so the ability to train mice on tasks also used in monkeys becomes critical. We trained mice on a modified random-dot motion (RDM) task, adapted from non-human primate studies (Britten et al., 1992), in which they discriminate between two directions of motion across varied levels of difficulty. This design exposes mice to varying levels of directional coherence, allowing us to measure behavioral effects following future experimental manipulations such as chemogenetic inhibition. Toward that goal, we developed an optimized training protocol for mice to perform RDM discrimination designed to maximize learning efficiency while minimizing stress. The protocol consists of sequential stages of training: habituation/acclimation, free reward, directional, dynamic, and maintenance, only advancing once a pre-defined accuracy threshold is reached. We trained 34 mice using this approach, and 80% of them learned to perform the task with the easiest condition in 40 training days. 16 mice completed the full protocol in 130 days. Our findings establish an efficient framework for training mice in complex perceptual tasks, which can be combined with neuroscientific tools to assess circuit function, allowing us to explore the evolutionarily conserved or divergent neural circuits underlying decision-making between mice and monkeys.

Comprehensive brain atlases are an instrumental prerequisite for neuroscientists, akin to geologic and topographic maps for geographers. In providing a spatial reference system, brain atlases allow for navigation to identified brain regions based on anatomical location. However, many standardized mammalian brain atlases have not been quantitatively validated for in vivo accuracy. Observations of various mouse brain atlases in use  reveal numerous inconsistencies and lead to unquantified errors in brain area targeting. My  hypothesis is that existing mouse brain atlases misrepresent real-world coordinates of the in vivo brain within the mouse skull. To test this, I am establishing reliable methods for the systematic measurement of true stereotaxic brain locations and quantification of coordinate discrepancies between the in vivo brain and atlases. Across a cohort of mouse subjects, I optimized the localization of fluorescent dye injections to quantify specific points in the in vivo space. I evaluated fluorescent dye injections using iontophoresis to control dye flow in mouse brain tissue through applied current. This achieved high-contrast fluorescence histochemical detection without diffusion into untargeted brain areas. In addition, I developed an algorithm that maps injection coordinates from histochemical imaging datasets and targeted stereotaxic brain locations to various brain atlas spaces. In our comparison of the average Euclidean distance between mapped real-world injection and targeted location coordinates across three standardized mouse brain atlases, we identified the MRI-based atlas to be the most accurate. Further, I computed and implemented non-negligible 3-dimensional affine transformations to correct discrepancies between the in vivo space and each mouse brain atlas. We expect this work to produce a validated and accurate coordinate system for targeting electrode insertions. This innovation will substantially improve the quality of large-scale data collection in labs around the world. 

Parkinson’s disease (PD) is a neurological disorder that affects patients’ movement, balance, and coordination, primarily due to the death of dopaminergic neurons. Traditionally, researchers use MPTP, a neurotoxin that destroys dopaminergic neurons, to replicate the motor symptoms of PD. However, this approach captures the later stages of the disease, making it difficult to develop early stage interventional treatment with this model. There is a long prodromal, or early, phase of PD, in which neuronal cells and circuits are changing before the neurons die and cause overt motor symptoms. A critical gap exists in our understanding of the early progression of PD due to the lack of robust primate models of this phase of the disease process. In an effort to create a prodromal phase model of PD, we made intrasnasal and intracranial injections of a pathological form of the protein alpha-synuclein (aSynPFFs) extracted from human PD patients, and used it in macaques. We quantified the motor changes in macaques using a modified version of Unified Parkinson’s Disease Rating Scale (UPDRS), which has 14 categories that each define a movement function of interest scaled from 0 to 3 (no symptom to highly impaired). To improve the detection of the changes, we used a deep learning software called DEEPLABCUT (DLC) to track the subtle motor changes seen in the macaques after exposure to aSynPFFs. By using quantitative approaches to assess motor function before and after aSynPFFs exposure, we hope to establish a timeline of neurodegeneration associated with PD in primates. Such a model would provide an important platform to assess therapies to halt neurodegeneration associated with PD.

Spinal cord injuries (SCI) affect over 1.2 million people in the United States, resulting in severe motor impairments due to disrupted communication between the brain and muscles. While physical therapy is the standard treatment of rehabilitation, its effect on recovery is limited. The pairing of spinal cord stimulation (SCS) and physical therapy is a promising new improvement for rehabilitation. SCS is thought to work by increasing spinal excitability, allowing more neural input to generate voluntary movement. However, preliminary data have shown that training on one task may interfere with progress in another, raising questions about the mechanisms underlying motor recovery after SCI and how to optimize rehabilitation strategies. In this project, we explore how multichannel, targeted, activity-based spinal stimulation (mTADSS) can enhance functional recovery in a rodent model of SCI. Using intraspinal stimulation, we examine whether training multiple tasks during a therapy period will interfere with the effect of recovery. Our experimental design consists of five groups of rats that first undergo baseline motor assessments, including training to evaluate grabbing ability and measure both grip force and range. Following these assessments, the rats receive a moderate cervical contusion injury, after which they undergo retraining with or without TADSS to assess its effects on motor recovery. I am responsible for operating the stimulation system and ensuring precise stimulation timing during physical training. I also collect and analyze behavioral and stimulation data to assess the impact of different rehabilitation approaches. Based on our preliminary data, we expect to find interference between tasks highlighting the need to develop better task training protocols to induce greater motor generalization. This research aims to contribute to the development of more effective rehabilitation strategies for individuals with SCI, potentially improving their mobility and quality of life.

Spinal cord injury (SCI) affects millions of people around the world, often leading to severe physical, psychological, and social consequences. Understanding how the brain and spinal cord react to injury is important for finding ways to help people recover lost movement. Previous research has investigated c-Fos expression, a protein that shows when nerve cells in the spinal cord are active, as a marker of neuronal activity in response to SCI; however, further investigation is needed to identify new pathways and technologies that could aid in the recovery of SCI patients. I am investigating how c-Fos behaves in the spinal cord of rats through Steve Perlmutter’s lab, part of the Department of Neurobiology and Biophysics at the University of Washington, which focuses on developing neuroprosthetic therapies - therapeutic interventions that restore lost neural function by electrical stimulation of sensory or motor pathways. These prosthetics enhance the nervous system’s ability to promote reorganization of brain and spinal cord connections, which can support improved motor recovery in conditions like stroke, traumatic brain injury, and SCI. In this study, I am investigating how c-Fos behaves in the spinal cord of rats before and after they are injured, and how different types of stimulation affect it. I use a technique called immunofluorescence to look closely at c-Fos activity in the lumbar and cervical areas of the spine, which are critical for motor control. The goal of this project is to further investigate which pathways in the spinal cord help recovery and how stimulation can affect c-Fos expression. Since the research is still ongoing, the study aims to contribute to the broader goal of improving SCI rehabilitation by providing insights into neuronal plasticity and supporting the development of new neuroprosthetic therapies to enhance motor function in SCI patients.

Hearing loss is a prevalent disability that is commonly caused by damaged hair cells, which are mechanosensory cells critical for hearing and balance. Among the ways in which hair cells can die—including aging, genetic predisposition, and noise exposure—is damage due to ototoxicity, which is when medications damage hair cells. Aminoglycosides, a commonly-used family of antibiotics, is known to cause hearing loss in patients that undergo multi-day treatments. Our lab and others have shown that certain aminoglycosides (e.g. neomycin) can cause acute hair cell death, whereas other aminoglycosides (e.g. gentamtcin) kill in a more delayed manner. In the case of delayed hair cell death, it has been shown that these aminoglycosides accumulate exclusively in the lysosome, which is the organelle that contains digestive enzymes. It is thought that a lysosomal stress response contributes to hair cell protection through calcium release and then the recruitment of lysosome-membrane-repairing proteins known as ESCRTIII. In my project, I use zebrafish live imaging to elucidate if ESCRTIII proteins are recruited onto lysosome membranes in aminoglycoside-treated hair cells. First, I create a transgenic zebrafish line containing IST1-GFP in its genome. IST1 is a part of the ESCRTIII complex and serves as a biomarker to track where ESCRTIII proteins are active in a cell. If the aforementioned hypothesis is true, then in aminoglycoside-exposed hair cells, we expect to see ESCRTIII proteins localized around lysosomal membranes following lysosomal stress response and calcium release. Elucidating the lysosomal repair mechanism in the context of aminoglycoside exposure is valuable for understanding how hair cells could survive ototoxic conditions. In the future, it may be possible to harness ESCRTIII proteins to prevent hearing loss induced by ototoxicity.

Innate immunity is the body’s first line of defense against pathogens, with macrophages and microglia playing key roles in combating infections in the olfactory epithelium (OE) and olfactory bulb (OB)/brain, respectively. Previous experiments in our lab showed that Influenza A virus (IAV) infection is limited to olfactory sensory neurons (OSNs) in the OE. However, how the virus interacts with the immune system in the OB—the region responsible for processing smells—and the rest of the brain remains unclear. Since the OE connects directly to the OB, which leads to deeper brain regions, this suggests a protective mechanism along this pathway that prevents the virus from spreading from the OE to the OB and further into the brain. In this project, we investigated the roles of microglia and macrophages in IAV infection using three mouse models: wild-type (C57), microglia-depleted (PLX5622-treated), and RAG1KO mice, which lack T and B cells and therefore adaptive immunity, the secondary defense of the immune system. We found that viral infection in the OE triggered significant macrophage activation, particularly when microglia were depleted. When microglia were absent in the brain, macrophages in the OE became overactive to prevent viral spread into the OB and brain, suggesting that microglia are crucial for immune activation in the brain. Analysis of Iba1+ cells (a marker for both microglia and macrophages) showed increased activation in response to IAV, with the PLX5622 and RAG1KO groups showing the strongest macrophage response. These findings highlight the role of macrophages in defending against IAV in the OE and suggest a complex interaction between immune cells in preventing viral spread along the OE-OB-brain pathway. Future analyses will explore responses of specific immune cells in the OB and brain, particularly in immunodeficient models, to better understand how the immune system combats viral infections.

Hearing loss affects approximately 40 million people in the US. It is primarily caused by the damage and loss of hair cells, which do not regenerate in humans. In the Raible lab, we use zebrafish as a model to study hair cell development, death, and regeneration. Unlike mammals, zebrafish can regenerate their hair cells after damage. I am currently using CRISPR-Cas9 gene editing technology to create mutant zebrafish to test a gene’s role in hair cell development and regeneration. We use guide RNA to target and mutate different genes that have been shown to be expressed in hair cells or support cells, which act as a new source of hair cells during regeneration. At 5 days post fertilization we quantify the number of hair cells and compare the numbers between mutant and non-mutant fish to test for developmental defects. If there are no defects, we treat these fish with the ototoxic antibiotic neomycin to kill their hair cells. After neomycin treatment, we wait 48 hours for the hair cells to regenerate and then compare the number of hair cells in non-mutant fish to mutant fish to examine whether the loss of that gene impacts hair cell regeneration. By developing an understanding of what genes are important for hair cell function and regeneration in zebrafish, we can begin to apply these findings to help with studies looking into hearing loss in humans.

Alterations to sleep structure have been observed in healthy aging humans as well as those diagnosed with Alzheimer’s disease (AD). To gain further insight into how sleep is affected by age and neurodegenerative diseases we will analyze sleep in healthy aged rats and in a transgenic rat model of AD. We collected neural data from the hippocampus of aged  (30-32 months old) and adult rats (4-9 months old) during 30-60 minute sleep sessions before and after the performance of a spatial navigation task. We have collected similar sleep data from transgenic F344AD rats (12 months old; a model of AD) and their wildtype littermates. First, we will combine movement tracking and measures of hippocampal local field potential (LFP) activity in the hippocampus to distinguish periods of awake activity, quiet wakefulness, slow-wave sleep, and REM sleep. Specifically, we will use an established measure, the theta-delta ratio, to distinguish slow-wave sleep from REM sleep. Using this approach, we will characterize the sleep structure of the young and old rats and the AD/control rats to determine if there are any differences in, for example, the amount of time spent in a particular sleep stage or the average length of each stage. In addition, we will investigate whether there are any differences in sleep patterns between shorter (30-60 minute) sleep sessions and longer (4 hour) sleep sessions. These analyses will determine whether our rat models of aging and AD recapitulate the sleep changes seen in aged humans with and without AD.

Retinal cell degeneration is one of the leading causes of blindness and vision loss caused by retinal diseases and is irreversible in humans. However, regeneration of retinal cells occurs after injury in some non-mammalian vertebrates and mimicking these strategies in humans could evolve treatment options for the visually impaired. Previous research in the Reh lab discovered a way to generate new neurons by reprogramming Müller glia (MG), a support cell of the retina, through overexpression of the proneural Ascl1 transcription factor in the mouse retina. To stimulate reprogramming, we used a lentiviral construct with a glial specific promoter (HES1) to drive the expression of ASCL1. However, HES1 represses its own expression by binding specific DNA sequences called N boxes which regulate gene transcription and expression, thus creating a negative feedback loop. In order to limit the negative feedback loop, we designed two new constructs using the HES1 promoter with modifications to the N box sequences. While the current construct has a reprogramming efficiency of approximately 25 percent, the aim of my project is to use constructs with modified N boxes to increase the ratio of MG reprogramming into neurons and verify specificity of the new constructs to MG cells. My research with mouse MG has shown that constructs with N box modifications significantly increase Ascl1 expression as compared to the construct with no modifications. These results seem promising and if reproducible, I will proceed with applying this strategy to human MG by using an in vitro culture system of retinal organoids.