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.    

In mammals, circadian rhythms are regulated by a hierarchy of oscillators governed by a central circadian pacemaker in the suprachiasmatic nucleus (SCN), which is principally entrained by the light-dark (LD) cycle. Recent experiments in our lab have revealed that cyclic 24-h fearful stimuli can act as a potent nonphotic zeitgeber, entraining circadian rhythms of behavior in mice and rats. This discovery utilized a naturalistic rodent cage with a safe nesting area separated from a foraging area where feeding and drinking occur. While foraging behaviors naturally occur at night, when the foraging area is rendered dangerous by nocturnal aversive stimuli (footshocks), animals entrain behaviors to the shock schedule by shifting activity to the daytime. Under conditions of fear-entrainment, SCN clock gene expression remains loyal to the LD cycle and the SCN is necessary but not sufficient for sustaining diurnal activity. Therefore, we propose the existence of extra-SCN fear-entrained oscillators capable of overriding SCN output and influencing behavioral timing. Here, we subjected 40 mice to either diurnal shocks (DS; control) or nocturnal shocks (NS) under a 12:12 LD cycle. Following confirmation of fear-entrainment, animals were released into constant conditions and sacrificed between 24-36h after the last presentation of footshocks, either CT 1 or CT13. Brains were dissected, sliced, prepared for immunohistochemistry processing, and c-FOS and PER2 protein quantification is currently underway in the SCN, basolateral amygdala, paraventricular nucleus of the thalamus, and dentate gyrus. We hypothesize that c-FOSs and PER2 expression within the SCN will align with the LD cycle, while centers involved in fear processing and memory will exhibit altered levels of c-FOS and PER2 expression in response to time-specific fear. Results from this study may be useful for identifying putative brain regions containing fear-entrainable oscillator(s).

In mammals, the primary mechanism regulating circadian rhythms is the central circadian pacemaker in the suprachiasmatic nucleus (SCN). The 24-hour light-dark (LD) cycle is the primary environmental cue, or zeitgeber, that entrains the SCN and sets its phase by adjusting its timing via phase advancing or delaying. Our laboratory has demonstrated that when mice are foraging outside of their safe nest, cyclic fearful stimuli can act as a nonphotic zeitgeber, entraining circadian rhythms and shifting activity from nocturnal to diurnal. However, the mechanisms of this so-called “fear entrainment” and phase-specific properties of cyclic fear remain unclear. This study examined whether cyclic fear via footshocks differentially entrains activity depending on the circadian phase of exposure. Mice were housed under a 12h:12h LD cycle and divided into three groups based on shock timing: the first six hours of the dark phase, the last six hours of the dark phase, and the middle of the light phase. Both dark phase groups showed delayed activity, with only the early dark phase group exhibiting evidence of entrainment. The mid-light phase group remained nocturnal. To further investigate the interaction between light and nonphotic entrainment, we conducted a follow-up experiment in which mice were placed under constant laboratory conditions (constant darkness) before undergoing cyclic footshock exposure. We hypothesized that, in the absence of light cues, the phase shifts induced by fear would differ from those observed under LD conditions, potentially revealing a distinct mode of nonphotic entrainment. Our findings so far suggest that entrainment to cyclic fear may only be achieved through delays, and that circadian oscillators may use different mechanisms of entrainment in response to photic vs. nonphotic zeitgebers.

Initial work from our lab has demonstrated that decision-making in OCD patients varies along the approach-avoidance behavioral axis depending on their response to deep brain stimulation (DBS) treatment. Patients who do not respond to DBS exhibit heightened risk aversion and consistently make avoidant decisions, while responders balance risk and reward in their decision-making process. In contrast, patients who receive excessive stimulation display disinhibited behavior, making choices that maximize reward regardless of potential risk. While this 2D task has provided valuable insights into approach-avoidance behavior in OCD, it does not fully capture the naturalistic behavioral responses observed in daily life. To address this limitation, we are developing a virtual reality (VR) task designed to quantify approach-avoidance behavior in a dynamic 3D environment. Participants complete probabilistic decision-making trials while wearing a VR headset, allowing for precise tracking of eye movements, hand positioning, and body dynamics. This project aims to provide a naturalistic task to analyze movement velocity and behavioral trends to identify neural biomarkers associated with approach-avoidance tendencies. This work will act as a stepping stone, enabling deeper insights into how neuromodulation regulates these behaviors. We are currently finalizing the development of this VR task and will demonstrate the feasibility of using this task to collect unique behavioral data on approach-avoidance behaviors. In the future we hope to use this task to identify biomarkers of approach-avoidance behavior and use that information to further refine neuromodulation treatment for psychiatric disorders.