The inner ear is crucial for both hearing and balance and undergoes rapid changes during embryonic, fetal, and postnatal stages. Its complex structure poses challenges in studying auditory systems, especially the organ of Corti responsible for sound perception. Hair cells (HC’s) within the cochlea are essential for hearing, and their loss in mammals results in permanent deafness due to their inability to regenerate. We recently established Early B cell factor 1 (Ebf1) as an important factor for the development of the cochlea. Our lab’s work with conditional knockout (cKO) mouse models revealed that Ebf1 restricts sensory development within the cochlear duct. The Slc26a9-Cre Ebf1cKO model deletes on embryonic day (E) 9.5. We have seen over 2-fold increases in HC’s and their associated support cells (SCs). We have developed a tamoxifen-inducible Sox2Cre recombinase mice model for precise timing of genetic manipulation within the cochlea. Specific temporal activation of tamoxifen-inducible Sox2Cre recombinase in the cochlea will uncover critical regulatory time periods for the establishment of the sensory domain. Activation of tamoxifen-inducible Sox2Cre at embryonic day 11/12 only shows an increase in inner hair cells. These findings lead us to ask, what’s the critical window for Ebf1’s regulatory role in cochlear development? To determine the optimal window for Ebf1's regulatory role, I will activate the Cre expressed in Sox2 Ebf1-cKO mice with tamoxifen at different embryonic days (9-14) via oral gavage. Immunostaining experiments utilizing HC markers (Myo7a), inner HC markers (Vglut3), and SC markers (Sox2) will be conducted. I will quantify HC densities and cochlear length of Sox2 Ebf1-cKO and littermate control mice. Due to tamoxifen toxicity, samples will consist of embryonic day E18 specimens. Anticipated results include varying HC numbers, innervation and the presence of ectopic sensory patches. This study will offer valuable insight into the temporal dynamics of Ebf1's regulatory role.

The retina is a unique neuronal structure in the eye that facilitates vision. Many diseases cause the death of retinal cells and this can lead to blindness. Frogs and fish have retinal stem cells that can repair the retina after retinal cell death; these stem cells are concentrated in a region called the ciliary marginal zone (CMZ). It was thought that humans lack these cells; however, we have discovered a region of the retina that has some features of the CMZ. We call this the Late Proliferative Zone (LPZ). One of my research goals was to determine whether the LPZ in humans also contains retinal stem cells that could be harnessed to repair the injured retinae. To start, I measured the area of small cuttings of fetal retinal tissue grown in culture, called retinospheres (RSs), over time and identified a window from 250-325 days gestation in which the LPZ of the human retina continues to grow after the rest of the retina is quiescent. This result shows that the cells of the LPZ can make new retinal cells much later than we thought, supporting the idea that these are retinal stem cells. My second goal was to find factors that can stimulate the growth of these cells. I tested several factors known to be important for the stem cells in frogs and fish. I found the effects of these factors on the types of neurons made by the LPZ. In sum, investigating different ways to manipulate the LPZ provides the field with insight into what is needed to regenerate cell types lost in blinding diseases.

By 2050, it is predicted that over half of the global population will be over the age of 65. Therefore, it is of the utmost importance that we study the impacts of aging on human health and well-being. One major obstacle researchers face in seeking to understand human health relates to the extensive number of newly discovered biological systems that exist and interact in every individual at various levels and timescales. Furthermore, many well-known age-related neurodegenerative diseases are thought to begin years to decades before any clinically relevant symptoms are present. The current study attempts to address some of these challenges by examining the interaction of cognition with immune function, inflammation, gene expression, and the microbiome. This research will allow us to begin unpacking the complex interactions of these numerous biological factors and their impact on natural aging and age-related neurodegenerative diseases. To accomplish these goals, our project has created and implemented a novel high-throughput cognitive testing system to assess a range of cognitive abilities including, but not limited to, attention, memory, and object recognition, on two species of well-known non-human primates (NHPs). At numerous time points, we concurrently collected and analyzed blood, fecal, and cerebrospinal fluid to characterize each animal's health profile. With our collection of data sets, we anticipate that older NHPs will display higher levels of neuroinflammation and decreased immune function. We hope to find correlations between these factors and other variables with genes that are known to be related to a host of neurodegenerative diseases including Alzheimer's Disease (AD), Alzheimer’s Disease-Related Dementia (ADRD), and Parkinson’s Disease, among others. Results from this ongoing project will unravel mechanisms associated with age-related neurodegenerative diseases, allowing for earlier detection; this early detection is regarded as the most effective approach for preventing and treating such diseases.

Pain is the number one reason why patients seek medical treatment, yet current pain therapeutics such as opioids have limited efficacy and produce harmful side effects. This has produced a critical need for the development of novel therapeutics for the treatment of acute and chronic pain. Cannabidiol (CBD) shows promise as an analgesic, but the mechanism of action is not well understood as it interacts with several receptors such as cannabinoid receptors CB1 and CB2 and noxious nociceptors TRPA1 and TRPV1. I am investigating how CBD acts on the nervous system to disrupt nociception (pain perception) utilizing the Danio rerio model system and human embryonic kidney cell line 293T (HEK 293T). I use behavioral assays with genetic knockout models to interrogate the molecular mechanism of CBD-mediated analgesia and ratiometric Fura-2 calcium imaging of HEK 293T cells that express TRPA1 or TRPV1 to further elucidate their responses to combinations of CBD, heat, and allyl isothiocynate (AITC, a TRPA1 agonist). My preliminary results indicate that CBD is pronociceptive at low concentrations (10uM) and analgesic at high concentrations (20uM). Recent experiments suggest that CBD’s pronociceptive properties occur via TRPA1 activation, and that this sensitization attenuates CBD-mediated analgesia. I anticipate each CBD receptor knockout will alter CBD-mediated analgesia, with CB1 and CB2 null animals experiencing deficits, while CBD-evoked analgesia may be potentiated in TRPA1 and TRPV1 null animals. I anticipate observing heightened intracellular calcium concentrations when HEK293T cells expressing TRPA1 are perfused with CBD, and increased responses to AITC when cells are perfused with CBD. Importantly, this project creates a platform for the investigation and characterization of minor cannabinoids and other potential therapeutics, using behavioral phenotype based screening to aid in the development of novel, non-opioid analgesics that can revolutionize pain treatment.

General anesthesia (GA) is administered as a sedative in nearly 60,000 surgeries daily in the United States. Yet, there is a very limited understanding about how GA impacts brain activity, leading to induced loss of consciousness and pain sensation. Preliminary work in the Heshmati lab has highlighted key subcortical structures that are engaged during anesthesia, but it remains unclear how activity in these regions and across the brain regulates awareness or pain sensation as anesthesia is induced (“induction”), maintained at a steady state (“maintenance”) and removed (“emergence”), as is done during surgeries. My work aims to identify the neural circuits that regulate the loss of consciousness and pain sensation during GA by recording local field potentials (LFP) from mice as they undergo volatile anesthetic isoflurane (ISO). During LFP recordings, I will insert small electrodes into highlighted regions of interest, to capture low-frequency extracellular voltage signals generated by the synchronized activity of nearby neural populations during the three periods of interest: induction, maintenance, and emergence from isoflurane GA. I will analyze the amplitude fluctuations and frequency patterns to identify synchronized oscillations within subregions and assess the level of synchrony, or coherence, across different regions. Given previous findings on the shared and opposed involvement of subcortical regions in pain and anesthesia, I expect to observe coherence among some of the regions, such as the amygdala and hypothalamus, but potentially anti-correlation within specific subsections, such as central vs. basolateral amygdala. Through these experiments, I will be able to monitor the effects of isoflurane anesthesia through a temporally-defined electrophysiological lens, capturing real-time activation dynamics of large neural populations across induction and recovery from anesthesia. Thus, my research aims to further develop our understanding of the brain under GA, by providing novel insight into the neural circuits regulating wakefulness and pain during surgical procedures.

Maladaptive aggression characterizes - or is comorbid with - many neuropsychiatric illnesses, and can have devastating effects on individuals, their caretakers, and healthcare professionals. Human aggression is typically demarcated as exhibiting either reactive (defensive) or appetitive (rewarding) components. Despite a significant clinical awareness of the differences between these aggression presentations, preclinical characterization of their relative circuitry and associated neuronal mechanisms are absent. Using recently established protocols within our lab, we are able to study and compare these aggression phenotypes in outbred male mice in a high throughput manner. Briefly, for appetitive aggression, we train mice to self-administer a novel subordinate intruder over 7 days using a trial design. In the reactive condition, we non-contingently administered intruders with the same frequency distribution as the appetitive mice. In the current experiment, we used CD1xVgat-Cre mice injected with pGP-AAV-syn-FLEX-jGCaMP7s in the rostral lateral septum (LSr) to examine cell-type specific activity via fiber photometry. GABAergic activity in the lateral septum has historically been implicated in the control of reactive aggression, but its role in appetitive aggression is unknown. My roles in this project include behavioral testing and filming of the mice, as well as scoring these videos for first attacks following intruder presentation. Using these timestamps, I will next analyze the changes in population level dynamics across different time points of aggression motivation, seeking, and consumption using the open source photometry analysis program GuPPy. We expect that the photometry results for mice in reactive and appetitive environments will show different patterns of activity, with more GABAergic activity during the consumption of reactive aggression. Interestingly, our preliminary results also show an increase in GABAergic activity when mice press the lever for a trial on which they subsequently attack, indicating that GABAergic activity may drive appetitive aggression seeking.

Social interactions in humans have shown to improve pain outcomes and diminish the development of mechanical hypersensitivity (allodynia) following injury. This effect is known as social buffering of pain; however, the underlying mechanisms are not well understood. Prior preclinical studies focused on forced social interactions between unfamiliar mice, lacking translational value to patients. To fill this gap, our research explores how volitional social behavior shifts pain sensitivity and affect following a neuropathic injury. Volitional interaction is key to socialization as individuals usually socialize because they want to, not due to force, which makes studying how mice voluntarily interact with each other important. To determine how volitional social interaction impacts both sensory and affective (emotional) components of pain, we use male and female mice who have received a spared nerve injury (SNI). Trained in social self-administration, mice learn to lever-press to engage with a familiar conspecific. Mice are then tested in von Frey where thin plastic filaments of increasing weights are applied to the mouse hind paw before and after SNI. These filaments do not cause pain, rather elicit a pain response of withdrawing the paw. To determine sensory sensitivity, the weight when the animal's paw is withdrawn is recorded as percent change from baseline. To determine changes in affective pain, the amount of time the animals hold their paw up, following withdrawal, is recorded as percent change from baseline. We found that male and female mice show significant attenuation in their mechanical hypersensitivity following volitional social interaction compared to mice deprived of volitional social interaction. Males show even less mechanical sensitivity, indicating that males may be more impacted by social analgesia than females. Understanding the divergent responses between male and female mice and the role of volitional social interaction in pain modulation, offers potential avenues for developing novel therapeutic strategies.

Neuropsychiatric disorders, such as major depressive disorder, pose a difficult challenge for healthcare providers. Treatments for such disorders vary in efficacy and come with detrimental costs. Historically, preclinical animal models have failed to incorporate the nuances of volitional human social behavior. This project used chronic social defeat stress in which mice experienced bouts of antagonistic encounters to induce depression-like behaviors in male and female mice, this was followed by self-administered social interactions within an experimental chamber in which lever presses were reinforced by social contact. The goal is to develop preclinical animal models that can be assessed to identify neural mechanisms responsible for stress-induced social motivation. Male and female mice will train to self-administer social interaction with a sex and age-matched housing partner over the course of ten 12-trial sessions. Next, experimental male and female mice will be subjected to both social and witness defeat (observation of social defeat) sessions followed by social self-administration. Before and after the 10-day social stress sessions, we will test social reward seeking via non-reinforced self-administration of social reward followed by a progressive ratio test. Brain tissue will be collected and prepared for immunohistochemistry and whole-brain clearing. Social defeat decreased social reward seeking behaviors in male mice. Witness defeat did not alter social reward seeking in males but increased seeking behavior in female mice. Social stress can be used to discern differences in social motivation in male and female mice as a result of stress-induced factors. There is potential in using whole-brain activity mapping to identify brain structures activated during social reward following social stress. We hope to build a technical tool for the field that can encompass whole-brain activity responsible for social stress responses by utilizing nuclear localization and retrograde tracing.

Recent advancements in the ability to measure and manipulate large-scale brain activity with high resolution have significantly enhanced our understanding of the coordination of brain-wide activity, a crucial aspect of brain computation. My project develops a novel system for simultaneously recording and inducing brain activity in mice, using calcium imaging and optogenetics to measure and manipulate brain activity, respectively. This system creates a more streamlined approach to measure activity across the entire cortex against stimuli and behavior, enabling comprehensive study into the mechanisms of inter-area brain activity. The experiments are done with conscious mice in a setup that includes custom software and hardware control using MATLAB, and data is analyzed using Python. One challenge is that the red optogenetic laser can cause aberrant visual responses. When the laser is near the retina, some of the light can travel through neural tissue and hit retinal neurons, ultimately starting a neuronal signaling cascade. An aberrant visual response can interfere with optogenetic effects. I have determined that laser powers greater than 1 mW can elicit this off-target visual response and that we can diminish the response to the laser using a noisy, flickering visual stimulus. When the flickering stimulus is present, the laser power needed to cause a visual response is increased by 100%. In the future, we will use the combination of techniques to understand what brain-wide mechanisms underlie goal-directed behaviors. By measuring and manipulating cortex-wide activity while a mouse completes a task, we can investigate communication between different parts of the brain and identify the mechanisms that impact learning. This technique has implications in more advanced studies of synaptic plasticity, computational modeling, and brain-wide cognition, offering promising new avenues for neuroscience research in the future.

Achieving consistent targeting of multiple simultaneous probes during electrophysiology experiments is a challenging and time-consuming process. Even with a planned insertion trajectory, experimenters still have to go through a lengthy process of positioning and inserting each probe. Electrophysiology experiments are increasingly focused on brain-wide coverage, requiring three or more simultaneous probes motivating researchers to accelerate their processes to reduce the duration of the experiment and the corresponding stress levels of their subjects. To improve the efficiency and reproducibility of multi-probe electrophysiology experiments, we developed two frameworks: a communication platform to allow software control of hardware micro-manipulators and an automation platform to perform multiple synchronous probe insertions. Each existing manipulator platform has proprietary software for programmatic control, which is rarely cross-platform and often exposes inconsistent interfaces. To standardize manipulator communication, we developed a Python server that acts as a generic cross-platform application programming interface (API). This platform ensures that client applications only need to interface with one API to be compatible with many different manipulator platforms connected across various computer operating systems. Building on top of this communication platform and an existing trajectory planning tool, Pinpoint, we next developed a system that automates the insertion process for multiple probes, saving time. The automation system provides three guarantees for researchers: first, that probes will reach their intended targets without manually introduced errors in targeting; second, that experiments can be repeated exactly to improve reproducibility; and third, that movement speeds are limited to low levels for reduced tissue damage. Because our software drives multiple probes simultaneously, complex multi-probe insertions are more manageable. Taken together, these open-source tools for communicating with hardware manipulators and automating multi-probe insertions enable the next generation of reproducible, high-efficiency, brain-wide electrophysiology data collection.

Typical data visualizations in neuroscience flatten 3D space into just two dimensions, limiting researchers ability to observe spatial relationships. To overcome this limitation, we have previously developed rendering tools to support exploratory 3D visualizations, specifically for neuroscience data. In this project, I am expanding the renderer to allow users to display and explore additional non-spatial dimensions of their data. These new tools will allow users to explore additional dimensions of their dataset such as time, stimulus properties, or the spatial position of an animal. For example, to explore time, I have developed an interactive slider bar that dynamically updates the 3D display and a corresponding linked 2D plot, providing a clear depiction of neural activity with relation to specific events. Scrolling along the 2D plot enables users to pinpoint their position in time relative to stimulus onset, with the 3D display concurrently adjusting to reflect the data from that specific snapshot in time. These functions are packaged into the API of the renderer, streamlining the process for users to transform raw data into intuitive and interactive visualizations. Reducing the complexity of the code expands the accessibility of these new features, making them more approachable for new users who may be less familiar with coding. By supporting additional dimensions, users will be able to develop visualizations that are tailored to their individual research projects. My objective is to create research tools that are versatile, applicable to a range of projects, and accessible to individuals with diverse levels of experience, including students and researchers of varying programming backgrounds.

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 remains 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 has 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: an evaluation of social self administration as an intervention for chronic pain, and histological analysis to identify gene expression changes as a result of social interaction. Future research will include mini-scope endomicroscopy recordings to visualize communication among major brain regions, and comparison of cell ensemble activity between groups of mice will lead to the identification of relevant neural ensembles and molecules.

The nervous system is composed of two major cell types, neurons and glia. While previously regarded as passive support cells for neurons, glia’s active roles in nervous system development and function have recently gained appreciation. Glia have elaborate cell shapes across which they asymmetrically localize neuron-regulatory proteins. Thus, to fully understand glial roles in nervous system dynamics, we must determine how glial morphology and polarity are regulated. To investigate this, we use the amphid sheath (AMsh) glia of Caenorhabditis elegans. AMsh glia exhibit apical-basal polarity, with apical-protein-marked membranes contacting neurons at the cell’s anterior, and basal membranes extending posteriorly toward the cell body. A striking feature of the apical membrane is a discrete projection within the anterior glial process, which we term the Glial Apical Boundary or “GAB”. We find that the GAB localizes many glial cues which regulate neuronal properties. Upon comparing GABs of different apical proteins expressed by a single cell, we discovered they all overlayed. However, GABs of bilateral glia can be out of register, suggesting that the GAB is independently localized on a cell-to-cell basis. Because AMsh glia derive from neuroepithelial progenitors, we then asked if mechanistic regulation of the GAB is analogous to that of epithelial apical domains. Surprisingly, canonical epithelial polarity regulators PAR-3 and PAR-6 do not localize to AMsh apical membranes. Furthermore, junctional markers AJM-1 and DLG-1, which demarcate epithelial apical-basal domains, are absent either from the GAB or from the cell altogether. RNAi knockdown of these and other polarity genes does not impact GAB integrity or morphology. Thus, the GAB is a novel polarity feature of AMsh glia not governed by canonical apical-basal polarity mechanisms. Our current work focuses on elucidating how the GAB develops and is maintained, with overall importance to understanding how glia localize regulatory proteins in health and disease.

The mammalian brain contains neurons and glia in equal numbers. Glia contribute to proper neuronal communication by removing unnecessary synapses via a process known as pruning. Pruning plays a critical role in brain development, learning, and memory. How do neurons communicate which synapses must be pruned by glia? One way is through cell surface exposure of the lipid phosphatidylserine (PS) which serves as an “eat me” signal to glia. In other contexts, like apoptosis, flippases and scramblase enzymes regulate PS exposure. Flippases are membrane transporters that restrict exposure of lipids like PS on the extracellular leaflet, while scramblases translocate lipids bidirectionally, thereby promoting PS exposure. We don’t know if these molecules also regulate PS exposure during glial pruning. The Singhvi Lab previously established conservation of glial pruning in C. elegans. This optically transparent model contains a stereotyped nervous system, making it ideal for studying in vivo pruning with single-cell resolution. We focus on a single neuron-glia pair, AFD-AMsh, and use widefield fluorescence microscopy and posthoc image analysis to quantify the number of neuron fragments pruned by glia. We previously found that mutants lacking the flippase TAT-1/ATP8A have more pruning, suggesting a novel inhibitory role for this protein. Here, I examine several candidate scramblases: SCRM-1/PLSCR1, ATG-9/ATG9, CED-8/XKR8, and ANOH-1/TMEM16F. I conduct genetic crosses to put mutants for these scramblases in a fluorescent background to visualize pruning and use the described methods to characterize any pruning defects. Specifically, I expect that relevant scramblase mutants will have less pruning, as the “eat me” signal is not properly exposed. Dysregulation of pruning contributes to neurodegenerative disorders like Alzheimer’s. Similarly, flippase and scramblase mutations are linked to human brain dysfunction. Thus, studying the role of these enzymes in pruning offers novel insight into human brain health and disease.