Within the field of anesthesia, the process of arousal following general anesthesia is still little understood. Of particular concern is the way a state of pain can modulate arousal, with the nucleus accumbens (NAc) as a brain region of interest as it serves many functions including controlling mood, pain states, and reward motivation. This study investigates how NAc principal cells change their firing during arousal and are influenced by a pre-existing pain state. To assess this, we leverage the partial sciatic nerve ligation model of chronic neuropathic pain to examine the influence of pain state on arousal. We then test groups of mice in their behavioral responses, including open field test, von Frey, hot plate test, and the return of righting reflex as a measure of arousal from isoflurane anesthesia-induced unconsciousness. In some mice, we record 1-photon calcium-related population activity in the NAc to analyze neural activity during arousal from anesthesia. Our findings will serve to illuminate the underlying brain circuitry involved in arousal from anesthesia and the influence of pain state, which can help improve anesthesia recovery and may reveal non-opioid or endogenous mechanisms for pain relief.

The ventral tegmental area (VTA) contains dopamine (DA) expressing neurons, which are critical for reward processing in the brain. DA neurons are tightly regulated by inhibitory GABA-expressing neurons; these GABA neurons have recently been found to be present in distinct subpopulations in the VTA. Opioids disrupt this regulation by inhibiting VTA GABA neurons via mu-opioid receptors (MORs), leading to increased DA activity and reinforcing drug-seeking behavior. However, the distribution of MORs across distinct VTA GABA subpopulations remains unclear. This study uses multiplexed in situ hybridization to map MOR (Oprm1) expression in genetically distinct GABA populations characterized by their expression of Pnoc, Crhbp, and Cbln4. Preliminary findings suggest differential Oprm1 expression, with Pnoc and Cbln4 populations showing high Oprm1 expression patterns, while Crhbp contains little Oprm1 expression. These results highlight the heterogeneity of VTA GABAergic neurons and provide insight into the mechanisms underlying opioid addiction, which may inform future therapeutic strategies.

The locus coeruleus (LC) is a major neuromodulator source with widespread projections to distinct functional targets that influence arousal, anxiety, learning, and other behavioral states. Our lab has previously shown LC excitation triggers the release of norepinephrine (NE) into the basolateral amygdala (BLA). Recent studies suggest LC terminal stimulation may release DA into the dorsal hippocampus (dCA1) enhancing novelty-associated spatial learning. Our recent data show LC stimulation evokes DA release. Previously, release across regions, paradigms, and behaviors typically associated with LC have not been characterized, due to difficulty in separating DA from NE using traditional sensing methods. Due to this, the relationship between the LC and other DA systems remains unclear. To understand the mechanisms by which the LC may release DA independently of the ventral tegmental area (VTA), a major DA source, we have employed optogenetic stimulation to evoke release from neuron terminals and quantify the release dynamics of NE and DA. We used fluorescent biosensors to detect NE and DA, captured by a fiber optic cable and amplified to observe the relative dynamics of DA release. These sensors have tuned affinity and selectivity for NE and DA and use fluorescence as a proxy for neuromodulator release. In this project, we aim to elucidate how and under what conditions the LC is releasing DA across regions with different functions during aversive and appetitive behaviors. These data will enhance our understanding of the LC neuromodulator signaling that can become maladaptive and afflict anxiety, addiction, and more, and also demonstrate that the release of DA from the LC is dependent on the behavior induced.

A physiological response to acute stress, called anxiety, is thought to be an adaptive feature that allows us to adjust our behavior to better approach the situation causing stress. However, in anxiety disorders this response is maladaptive, leading to excessive anxiety. A key neural circuit is the projection from the locus coeruleus (LC) to the basolateral amygdala (BLA), and activation of this circuit produces anxiety-like behavior. However, little is known about how this alters the activity of BLA neurons. My Mary Gates research project seeks to utilize machine learning to understand how neuromodulatory input from the LC to the BLA alters the correlated activity of BLA neurons and their encoding of anxiety-like behavior. Mice expressing the excitatory opsin ChrimsonR in the LC and the calcium indicator GCaMP6s in the BLA received tonic (5hz) stimulation of LC terminals within the BLA through a GRIN lens to mimic stress-like release of norepinephrine into the BLA. LC terminals were stimulated while recording individual BLA neuron activity during a conflict-based test of anxiety-like behavior, the Elevated Zero Maze (EZM). To evaluate the correlated activity of BLA neurons as a function of stimulation, I used caGraph, a Python package that utilizes graph theory approaches to test the correlation of neurons from calcium imaging data. I investigated how stimulation affects graph theory communities (densely connected clusters) and clustering coefficients (strength of clustering) and found that stimulation causes an increase in the clustering of BLA neurons. To test the functional consequence of these ensemble shifts, I am using classification algorithms to assess the population encoding of the BLA neurons. I expect that stimulation of the LC terminals will increase the encoding of anxiety-like behavior. The findings of this project deepen our understanding of how the LC-BLA circuit mediates anxiety-like behavior, and may uncover novel treatment strategies.

Dopaminergic signaling within the striatum plays a crucial role in modulating reward and aversion, shaping behaviors such as food-seeking and consumption. While striatal dopamine release has been implicated in reinforcement learning and decision-making, the spatial and temporal dynamics of dopaminergic activity along the anterior-posterior axis of the striatum during consummatory behavior remain poorly understood. We investigated the role of dopamine in the striatum during the consumption of multiple solutions by employing a trial-based multi-spout behavioral paradigm with head fixed mice. To record the dopamine activity in the ventral and dorsal striatum, we utilized multi-site fiber photometry to record the fluorescent biosensor GRAB-DA2m along the anterior-posterior axis. Food restricted mice were given varying concentrations of sucrose as rewarding stimuli, while water restricted mice were given varying concentrations of sodium chloride as aversive stimuli. Our results revealed that dopamine responses scaled more across concentrations in the anterior regions of the striatum compared to the posterior regions. Additionally, we found more distinction between dopamine responses for the various concentrations of the aversive solution compared to the rewarding solution. Lastly, posterior striatal dopamine responses had a more rapid onset upon stimulus consumption, whereas anterior regions exhibited delayed responses, highlighting region-specific temporal differences in dopaminergic encoding. These findings refine our understanding of dopaminergic circuitry within the striatum and how dopamine-mediated responses to rewarding and aversive stimuli regulate feeding behaviors. By exploring this pathway, we offer potential insights into the mechanisms underlying disorders characterized by dysregulated reward including eating disorders and obesity.    

Cardiac-specific promoters, such as NK2 homeobox 5 (NKX2.5), are essential for driving gene expression during early heart development, making them valuable for studying neonatal congenital heart diseases. However, the large size of the NKX2.5 promoter limits its use in adeno-associated virus (AAV) delivery systems, restricting vector space for therapeutic genes. This study aims to develop a compact hybrid NKX2.5 promoter that retains cardiac specificity while enhancing its efficiency in early-stage cardiac research and gene therapy. To achieve this, we replace the enhancer region of NKX2.5 with a shortened cytomegalovirus (CMV)-derived enhancer, preserving cardiac specificity while reducing promoter size. The hybrid promoter is then cloned into an AAV vector to drive green fluorescent protein (GFP) expression for assessing transcriptional activity and tissue specificity. Following AAV injection into pregnant mice, we will harvest early-stage embryos to evaluate GFP expression in cardiac tissues, comparing the hybrid promoter’s efficiency against the original NKX2.5-driven GFP expression. This study addresses current limitations of cardiac-specific promoters by developing a streamlined version optimized for gene delivery in neonatal models. Our findings strives to enhance gene therapy strategies for congenital heart diseases and provide insights into early cardiac gene regulation.

Mechanistic target of rapamycin (mTOR) functions in a protein complex with raptor to control protein synthesis in eukaryotes. A reduction of function mutation in C. elegans raptor is resistant to hypoxic death. This mutation, a missense at amino acid 1033 in the daf-15 gene, is interesting because the mutation site is conserved in all mammals, suggesting that this work could shed light on hypoxic injury mechanisms in humans. The Crowder lab has discovered that a mutation called tm11331 in a gene involved in purine metabolism blocks the hypoxia resistance of the raptor mutation. We hypothesized that the tm11331 mutation restores normal protein synthesis to the raptor mutant and therefore restores hypoxic sensitivity. For my project, I examined this hypothesis by measuring nucleolus size as an indirect measurement of protein synthesis. Four strains were used in this assay: unmutated (wild-type) worms, worms with the raptor mutation, worms with the tm11331 mutation, and worms with both raptor and tm11331 mutations. From previous experiments, we know that raptor mutants have smaller nucleoli than wild-type worms, indicating that protein synthesis rates are lowered in mutated worms. We would therefore expect that protein synthesis rates and nucleolus size would be restored in worms made hypoxia sensitive by the addition of tm11331. For this assay, all strains contained a fluorescent protein that labelled the nucleoli, allowing me to image nucleoli under fluorescence. I processed each image and measured average nucleolus size in worms from each strain. Our data shows that the tm11331 mutation increased nucleolus size in strains both with and without raptor mutation. In fact, the combination of tm11331 and the raptor mutation was not significantly different from wild type. Thus, our data supports the hypothesis that the tm11331 mutation restores hypoxic sensitivity by normalizing protein synthesis.

Strokes and heart attacks caused by a lack of oxygen, called hypoxia, are among the most prevalent form of debilitating diseases in the United States. Hypoxia has been shown to cause hypoxia-induced-fragmentation of the mitochondria altering their size, shape, and distribution (known as the mitochondrial dynamics). However, to what extent these dynamics are involved in hypoxic cell death remains unestablished. The Crowder lab through a C. elegans mutagenesis screen discovered a reduction-of-function mutation in rapTOR that confers strong hypoxia resistance. rapTOR functions in a complex with mTORC1 to control cellular metabolism including mitochondrial function. We decided to investigate whether the hypoxia resistance of the rapTOR mutant is from alterations of mitochondrial dynamics in response to hypoxic injury. To measure the mitochondrial dynamics, I visualized the mitochondria with an outer membrane fluorescent protein, in wild type and mutant worms with and without hypoxic exposure. I analyzed the images blinded to their genotype and hypoxic condition and scored mitochondria as primarily fragmented or tubular, which served as a surrogate for detecting changes in mitochondrial dynamics. For a more quantitative analysis, I utilized image processing MATLAB code and determined differences in images using principal component analysis. My analysis showed hypoxia induces small, rounded mitochondria in C. elegans resembling mitochondrial fission. I found the mitochondria in the rapTOR mutant displayed decreased hypoxia-induced-fragmentation after hypoxia. Then when I combined the rapTOR mutant with a hyperfragmented mitochondria mutant it showed fragmented mitochondria with and without hypoxic exposure. However, the double mutant is also hypoxia resistant, which is not consistent with our hypothesis that mitochondrial fragmentation drives hypoxic cell death. Therefore, we reject our hypothesis and conclude that rapTOR is hypoxia resistant from a mechanism distinct from that controlling mitochondrial fission.

Despite the widespread use of general anesthesia, our understanding of mechanisms by which anesthetics and analgesics induce unconsciousness remains limited. This study used a transgenic mouse model (FosTRAP2) to investigate neural circuits that are active during isoflurane-induced anesthesia. FosTRAP2 mice were retro-orbitally injected with an AAV-PHP.eB virus expressing Cre-conditional DREADDs (designer receptors engineered to be activated by designer drugs), which was followed by general anesthesia exposure, where 4-hydroxytamoxifen was injected to chemogenetically label isoflurane-activated cells brain-wide with DREADDs. We subsequently implanted the mice with wireless mechano-acoustic (MA) devices to record peripheral physiologic data such as heart rate, respiratory rate, temperature, and physical activity. To determine the functional impact of isoflurane-activated circuits, chemogenetic reactivation was induced via clozapine-N-oxide (CNO) injection, with concurrent video recording for pose estimation with Social LEAP Estimates Animal Poses (SLEAP), and behavior classification with Simple Behavioral Analysis (SimBA). Combining both physiologic data and machine learning provided us with an experiment pipeline that allowed us to better study brainwide isoflurane-activated neural ensembles. We found that reactivation of these circuits led to a significant reduction in heart rate, body temperature, physical activity, accompanied by a reduction in typical active behaviors, such as grooming and rearing. By gaining a deeper understanding of how general anesthetics alter neural circuits, we hope to uncover the complex relationships between brain activity and consciousness, with important implications for improving anesthetic practices and developing novel sedatives or analgesics in the future.

The current understanding of mechanisms of anesthesia and the function of anesthesia-activated brainwide neural circuitry is very limited. Additionally, there is an urgent need to develop new non-opioid analgesic drugs, and targeting anesthesia neural circuitry could provide a novel path to pain relief. To investigate the function of this circuitry, we use a brainwide approach to perform chemogenetic manipulations in a FosTRAP2 transgenic mouse model. Briefly, FosTRAP2 (Fos-2a-Cre) mice receive retroorbital injections of a Cre-dependent virus expressing chemogenetic DREADDs. Mice then undergo isoflurane anesthesia exposure and midway through the exposure, they receive an intraperitoneal injection of 4-hydroxytamoxifen to induce activity-dependent chemogenetic labeling of isoflurane-activated brainwide ensembles. I use behavioral analysis pipelines to analyze how the activation of these ensembles affects thermal nociceptive processing after mice are induced into a lightly anesthetized state and subjected to analgesia testing. Mice underwent the warm water tail withdrawal and hot plate assays. I then use a combination of manual annotation and pose estimation approaches with supervised machine learning using Social LEAP Estimates Animal Poses (SLEAP) followed by Simple Behavioral Analysis (SimBA) to provide insight into behavioral signatures and classifications. I identify a number of occurrences for behaviors such as tail withdrawal, latency to jump, and paw grooming, which is used to infer thermal anti-nociception in open field testing. I also helped develop five distinct behavioral classifiers: rearing, grooming, freezing, circling, and Straub tail response. With the resulting behavioral analysis, I can investigate how targeting brainwide anesthesia-activated neural ensembles produces anti-nociception. Anesthesia, although used for many common procedures, is not widely available to the general public and must be administered by a medical professional. Understanding the mechanisms behind its effect on pain processing is a gateway for revolutionary research that could potentially eliminate the need for opioid medications in the future.

Sleep disruption is a key factor that contributes to cycles of relapse in opioid addiction, a pressing public health concern. However, the underlying mechanisms of this sleep disruption are yet to be well-understood. Gaining a better understanding of the neural circuits involved in opioid-mediated sleep disruption may help to develop new treatment to mitigate the risk of opioid dependence. We previously found in a mouse model of escalating morphine administration and withdrawal that the locus coeruleus (LC), the brain’s primary source of cortical noradrenergic projections, is hyperactive during opioid withdrawal and is accompanied by a shift in sleep pattern during the first withdrawal day. However, its precise role in affecting opioid-mediated sleep disruption remains unclear. We are now investigating the role of LC noradrenergic projection to the paraventricular thalamus (PVT) in opioid-induced sleep disturbances, as the PVT is known to promote wakefulness. As PVT manipulation has previously been shown to reduce opioid-induced sleep disruption, I am examining the contribution of the LC-PVT circuit in this disruption in sleep pattern. To address this question, I am quantifying LC projections to the PVT by clearing brain tissue in mice expressing a fluorescently tagged synaptic marker in LC neurons, and counting the number of LC synapses in the PVT. Additionally, we  quantified norepinephrine release in the PVT using a genetically encoded fluorescent sensor and fiber photometry, and measured cortical activity using electroencephalogram (EEG). Our preliminary data show a potential decrease in NE activity during the first withdrawal day, but more mice are needed for this experiment. Additionally, given the elevated LC activity on the first withdrawal day, we plan to inhibit LC-PVT circuit on that day to determine if sleep disruption can be prevented. Together, these studies will help better define how changes in the noradrenergic circuits contribute to sleep disruption from opioid use.

Disruptions in the mechanisms underlying threat detection and maladaptive aggressive behaviors are core features of several psychiatric disorders, including anxiety disorders and post-traumatic stress disorder (PTSD). In these conditions, heightened vigilance and attentional biases toward perceived threats can contribute to inappropriate aggression or avoidance behaviors, underscoring the need to understand the neural mechanisms mediating threat assessment and aggressive responses. We aim to better understand threat assessment and responding by interrogating brain region and cell-type specific activity patterns during unfamiliar social encounters using mice as our model system. Recent studies have identified the posterior paraventricular thalamus (pPVT) as a hub for processing sensory and emotional information in response to stress, predators, and other aversive contexts to facilitate a choice action. Despite its relevance, the role of pPVT in social-emotional brain circuit function remains unexplored. Recent transcriptomic datasets have revealed genetically identifiable clusters within PVT, specifically highlighting estrogen receptor-1 (Esr1) as a genetic marker for more posterior areas of PVT. In our behavior paradigm, mice with intact pPVT^Esr1 neural activity selectively attack novel conspecifics with unfamiliar traits (out-group) but not those with familiar traits (in-group) when introduced into their home cage. Given this, we designed an experiment using chemogenetics, a technique that utilizes genetically engineered receptors (DREADDs) to modulate neural activity, to test the involvement of pPVT^Esr1 neurons during unfamiliar social encounters. We have found that selectively inhibiting pPVT^Esr1 neurons using Gi-DREADDs reverses attack behavior, suggesting a putative role for these neurons during threat assessment and response processes. To follow up on these results, we are selectively exciting these neurons using Gq-DREADDs to determine how increased excitatory activity within pPVT^Esr1 neurons affects aggressive behaviors towards in-group and out-group intruders. We hypothesize that chemogenetic excitation of pPVT^Esr1 neurons will increase aggressive behaviors toward intruder mice for the entirety of the trial.

The sensation of acute pain is fundamental to survival, indicating tissue damage that motivates an animal to engage in adaptive protective behaviors. Chronic pain, however, is persistent pain beyond typical recovery window and serves little adaptive function. The negative emotional component inherent in chronic pain contributes to the development of comorbid psychiatric disorders such as depression, social aggression, and social withdrawal. Our research aims to understand the bidirectional relationship between pain and social behavior, by evaluating mechanical sensitivity and changes in social motivation, reward, and interaction following a neuropathic injury. Using social self-administration (SSA), pair-housed mice were placed in operant chambers and underwent voluntary lever press trials for the reward of social interaction with their cage mate. Mice also underwent mechanical hypersensitivity response assays called von Frey where increasing weights of plastic filament were applied to the hind paw. Following baseline von Frey testing and the acquisition of the SSA task, mice then received a spared nerve injury (SNI) to induce neuropathic pain. After surgery recovery, mice were returned to the lever press and von Frey trials at different post-operative windows. Pain sensitivity was determined by the filament weight in which the animal withdrew their paw during von Frey. Changes in social behavior were measured via changes in lever press frequency and interactions during trials. Behavior changes were quantified using Simple Behavior Analysis (SimBA) machine learning to classify interactions during social trials. Once the trials were completed, brain tissue from regions associated with reward and social neural circuitry was collected and investigated using transcriptomic methods. Our data found sexually divergent social adaptations and gene expression following chronic pain. Future experiments will further delineate these sex-specific adaptations following a traumatic injury. This research can inform social intervention as an adjunct or alternative treatment to pharmacological pain intervention and its comorbidities.

Dissociation is an altered brain state caused by trauma, epilepsy, and drugs in which critical mental functions such as sensory processing and consciousness are disconnected. While its brain circuit mechanisms remain underexplored, their improved understanding would not only help treat dissociative psychiatric disorders but also help develop more precise dissociative anesthetics. Here, we used immunohistochemistry, in vivo fiber photometry, high-density electrode recordings, and machine learning behavioral analysis in a ketamine-induced dissociation model in mice to address how affective processing is disrupted in dissociation. We hypothesized that ketamine-induced dissociation alters the signaling of the locus coeruleus-norepinephrine (LC-NE) system, a brain region responsible for regulating stress and aversive responses. Previous studies have found that suspending a mouse by its tail induces escape-related behaviors, which are lost when treated with a dissociative dose of ketamine (50-100 mg/kg). We performed tail suspension experiments to further characterize these behavioral differences using a machine learning approach. Using DeepLabCut, a deep learning algorithm to track animal body parts, and Keypoint Moseq, an unsupervised machine learning algorithm for decomposing behavior into behavioral modules, we characterized differences in these behavioral modules between awake and dissociated states. We then compared changes in LC activity between ketamine- and saline-treated mice, using cFOS immunostaining as a marker for neuronal activation. We also used fiber photometry to examine downstream norepinephrine (NE) release in the medial thalamus during tail suspension, using a genetically encoded fluorescent NE sensor to measure NE activity in real-time. Our data shows increased NE activity during tail suspension, suggesting that the LC remains responsive in the dissociative state. This responsiveness may suggest that the disruption of the aversive response in dissociation is downstream of the LC-NE system. We plan to examine the medial prefrontal cortex and basolateral amygdala as possible loci of disruption using fiber photometry, Neuropixel recordings, and optogenetics.