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.

Despite recent advances in monoclonal antibody (mAb) technology and its rapidly growing market share, therapeutic targets for mAbs are currently limited to membrane proteins which consist of up to 30% of total proteins encoded by the human genome. The other 70% of cytosolic protein targets remain inaccessible inside the cell. Thus, research into intracellular protein delivery is critical to unleash the full potential of protein therapeutics. For example, mAbs can target oncogenes, enzymes, and the complex signaling cascades within the cell, unlocking a completely new domain of protein targets. Current methods for intracellular protein delivery involve either low protein throughput with minimal cell damage/cytotoxicity or high throughput approaches that compromise cell viability. The Gao lab recently developed a highly efficient technology that allows small proteins to be directly delivered into the cytoplasm with minimal damage to the cell, by cholesterol tag. To further this research, we developed a new version of the tag via the covalent linkage of Coomassie Blue dye with 2-hexyldecanoic acid, branched alkyl chains. This new tag could deliver mAbs, specifically immunoglobulin G (IgG), labeled with fluorescent dye. Through this project, I (i) carried out organic synthesis of the new tag, (ii) delivered secondary antibody into HeLa cells, (iii) confirmed protein internalization through fluorescent microscopy, and (iv) delivered anti-Vimentin primary antibodies for live cell imaging of intermediate filament. Ultimately these four aims demonstrate successful intracellular mAb delivery while maintaining its native protein structure. This allows us to utilize this technology to deliver protein therapeutics targeting all kinds of cytosolic proteins including oncogenic proteins such as p53, RAS, and MYC.

CD-1 outbred mice are a preferred mouse model used in behavioral research due to their genetic diversity and overall capacity for acquiring learned behavior. Psychiatric traits involve cognitive brain function, and psychiatric disorders always result in cognitive dysfunction. Therefore, behavioral research aimed to model psychiatric disease states can maximize translational value to human brain function and behavior by incorporating the study of effects on cognition in CD-1 mice, specifically. However, behavioral tests that target cognition sensitivity have been only made in other mouse strains. Here, we present a study establishing a human-compatible touchscreen-testing environment where we conduct the 5-choice Serial Reaction Time (5-CSRT) task, a mouse-friendly adaptation of the human continuous performance task on CD-1 mice from both sexes to phenotype baseline attention, impulsivity, and motivation. The 5-CSRT task is administered within automated Bussey touchscreen chambers and requires extensive behavioral shaping, habituation, tone, and screen touch training sessions designed to activate underlying neural circuitry involved in cognitive function. Before moving on to the next session, specific criteria must be met in order to promote acquired learning. These sessions become progressively more difficult, with an ultimate goal of priming the mice for the 5-CSRT touchscreen task, whereby animals need to find and touch a visual target being displayed from 5 available options on a touchscreen within a given amount of time to receive a food reward. To analyze and phenotype cognition, we combined the use of camera recordings to observe high-resolution behavioral dynamics in addition to summary task metrics that include accuracy, omissions, premature responses, and food-retrieval latency. Our results establish the use of CD-1 mice with human-compatible touchscreen-based approaches to facilitate an entry point in the effective modeling of psychiatric disorders that can map to human cognitive dysfunction.

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.

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.

Multitasking, such as walking and talking, is common for humans and other animals, yet animals are limited in how many behaviors they can perform simultaneously. The neural circuit mechanisms that limit multitasking are not well understood. Uncovering these mechanisms will help us understand how brains seamlessly combine behaviors. In turn, this will improve our understanding of some disorders, such as Parkinson’s Disease, wherein certain symptoms, like difficulties with walking and talking, are exacerbated during multitasking. I am investigating how neural circuits for known behaviors interact to limit multitasking in a genetic model system, the fruit fly Drosophila melanogaster. During the first year of my project, I developed a rig to record complex behaviors and I added optogenetic activation to the rig in order to activate neurons that drive one motor program during single and multitasking contexts. I am now preparing to conduct experiments with this rig, comparing the probability that a fly extends a wing (driven via optogenetic stimulation) while resting (single-tasking) vs. walking (multitasking). I expect that a fly will have a higher wing extension probability while single-tasking than while multitasking. The results of my project will help uncover how neural circuit interactions shape an animal’s ability to multitask.

What is personal space? It describes the “invisible bubble” immediately around an individual, where intrusion by others may feel uncomfortable or even threatening. Despite personal space being important for determining the size and dynamics of large groups and how animals behave, it is unknown how animals generate and maintain their sense of personal space. I explore this question using Drosophila flies, a powerful model system for studying behavior. Individual flies communicate via a large set of behaviors (e.g. kicking, flicking the wings, flying, and running) that can shape group interactions. Between pairs of flies, there is a rich repertoire of interactions that cause them to either move away (e.g. appendage touching) or move closer (e.g.courtship behaviors). To capture these behaviors, I use high resolution cameras running at 150 fps to collect videos of flies interacting in a circular arena and deep-learning software for annotating body parts (SLEAP). I use this data to track the poses of multiple flies throughout time and quantify appendage touching behaviors in addition to an estimate of each fly’s personal space. Using optogenetics, I model a situation in which a wild-type fly responds and adapts to being surrounded by other flies that crowd tightly. One expected outcome is that the wild-type fly will increase appendage touching in response to the space around itself shrinking. My goal is to determine exactly how the dynamics of such inter-fly interactions form a sense of personal space for each fly. My work will uncover the patterns of interactions that develop personal space and how these patterns scale out to larger social networks.

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.

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.

Pulmonary Arterial Hypertension (PAH) is a deadly vascular disease, affecting the blood vessels of the lungs rather than the systemic circulation, with no existing cure. PAH is characterized by pulmonary arterial smooth muscle cell (PASMC) hypertrophy and hyperplasia, which increases resistance to blood flow within the pulmonary arteries and leads to rapid symptom progression and death from right heart failure over several years. We hypothesize that defects in PASMC differentiation and alignment may contribute to PAH. Prior work has shown that micropatterned scaffolds encourage vascular SMC alignment and differentiation towards a contractile phenotype. To test whether these responses differ in patients with PAH, we designed a micropatterned collagen scaffold atop a glass coverslip. Scaffoldings were imprinted with either alternating 10-µm wide x 10-µm deep microchannels or left unpatterned. Explanted PASMCs from patients with PAH or failed donors (controls) were cultured on patterned versus unpatterned constructs and alignment, protein expression, and cellular morphology were compared across conditions. I evaluated 3 PAH and 3 control subjects and have collected preliminary data for each condition (control versus PAH), with three technical replicates each. Through these preliminary studies, I have demonstrated success of my model with consistent alignment observed on patterned substrates. Excitingly, PASMCs from patients with PAH expressed significantly decreased levels of the contractile protein, Calponin, when compared with control cells, including after responding to cues that promote alignment and contractility. This suggests that PAH PASMCs remain in an inappropriately synthetic or proliferative state. Subsequent testing will include assessment of calcium signaling in response to contractile stimuli and transcriptomic evaluation of cellular responses to micropatterning. This work will enhance understanding of whether SMC abnormalities contribute to disease initiation and progression in PAH and will contribute to the broader effort of developing more complex models of pulmonary vascular disease.