Dopamine (DA) producing neurons of the ventral tegmental area (VTA) in the midbrain regulate reward association learning and motivation. These DA neurons are modulated by neuropeptides and can be separated into distinct subpopulations based on differential gene expression, regulation of activity, and projection patterns. But how these different patterns are established and contribute to distinct functions of DA subpopulations remain poorly understood. One potential key component for these neuropeptides is the transient receptor potential canonical (TRPC) channels. Specifically, we identified the gene encoding TRPC type 6 channel (Trpc6) as having enriched expression in the VTA DA neurons. To determine whether Trpc6 is differentially expressed in VTA DA subpopulations, I utilized the quantitative, multiplexed in situ hybridization methods. Using wild-type mice, I probed for the expression of tyrosine hydroxylase (Th), a marker of all DA neurons, and Trpc6 as well as markers of two subpopulations, corticotropin releasing hormone receptor 1 (Crhr1) and cholecystokinin (Cck). The analysis showed that Trpc6 expression is significantly higher in the Crhr1 subpopulation, of 81%, than in the Cck subpopulation, of 66%. Because neuropeptides like neurotensin increase calcium concentration in DA neurons, we hypothesized that TRPC6 contributes to these neuropeptide-evoked calcium signals. To investigate the role of TRPC6 in DA signaling, I used a viral-based CRISPR/Cas9 approach to induce selective mutagenesis of TRPC6 in specific DA subpopulations. Then, I assessed the calcium responses of subpopulations to neurotensin by measuring the amplitude and neurotensin-evoked oscillations using acute brain slices. We expect the calcium responses to decrease more in the Crhr1 subpopulation than in the Cck subpopulation compared to the control as the Crhr1 population has higher Trpc6 expression. By elucidating the role of TRPC6, we hope to contribute to discovering pharmacological interventions for diseases caused by dopaminergic system dysfunctions such as Parkinson’s disease and substance use disorders.

The peptide neurotensin (NTS) has been known as a regulator of dopamine neuron activity and its system, which modulates numerous functions in the brain. Although ample research has now demonstrated that NTS in Ventral Tegmental Area (VTA) increases dopamine release in some regions, much remains unknown about the endogenous sources of NTS in the VTA and the impact of physiological NTS release. Recent NTS mapping data from the Soden lab demonstrated that there is a NTS projection from Periaqueductal Gray (PAG) to the VTA and also to hindbrain regions including the ventral medulla. This project investigates the effect of this interconnection between these three regions on the dopamine system. Utilizing advanced techniques in circuit mapping, mice will be injected in the VTA and the ventral medulla with fluorescent Retrobeads or a retrograde virus (rAAV2 anti mcherry or GFP), which are taken up by synaptic terminals and migrate up the axon retrogradely to label cell bodies, one color assigned for each region. Following euthanasia, mice brains will undergo immunohistochemistry such as histology and in-situ RNA staining. Then, data will be collected using imaging microscopes for results and further analysis. If NTS neurons in the PAG have green and red expressions, this will indicate that the same population of neurons send axons to both downstream regions, compared to PAG NTS neurons with only one color, indicating the presence of two separate neuron populations. Experiments on retrograde mapping of NTS inputs will contribute to building onto our current knowledge about VTA-PAG-ventral medulla circuit and effects on dopamine neurons following their interplay. In the end, our goal is to establish a novel understanding of endogenous NTS signaling mechanisms, mediation of complex reward processes, and treatment targets with experimental outcomes, giving rise to the development of therapeutic interventions towards addiction and related psychiatric disorders.

Understanding the complex neural mechanisms underlying motivated behavior and reward processing is crucial for advancing our knowledge of addiction and related disorders. In this study, we explore the role of neurotensin (NTS) peptides in modulating dopamine neurons within the ventral tegmental area (VTA), a key brain region implicated in reward processing. Previous research has highlighted the reinforcing effects of optogenetic stimulation of NTS inputs from the lateral hypothalamus to the VTA. However, a gap remains in understanding how NTS signaling interacts with other brain regions involved in motivated behavior. Our research addresses this gap by investigating the contribution of NTS and fast neurotransmitters in circuit-specific behavioral activation within the VTA. We hypothesize that distinct NTS inputs differentially influence motivated behavior in a combined NTS and GABA/glutamate-dependent manner through optogenetics and circuit mapping. We employ a multidisciplinary approach combining optogenetics, CRISPR gene editing, and behavioral assays to achieve this. Specifically, I utilize optogenetic stimulation to selectively activate NTS inputs from diverse brain regions, including the periaqueductal gray (PAG) and the pedunculopontine tegmentum (PPTg), targeting specific input regions via unilateral injections of AAV1-FLEX-Chrimson-mCherry. Control mice receive AAV1-FLEX-mCherry alone to ensure the specificity of our manipulations. Following surgical procedures and acclimation to the experimental setup, I conduct behavioral assays on mice, specifically Real-Time Place Preference (RTPP) and open-field testing to assess the effects of optogenetic stimulation on locomotion and anxiety-related behaviors. Subsequently, we investigate the mediating role of NTS and neurotransmitters by employing CRISPR viruses to target genes related to different components of neurotransmission. By integrating cutting-edge techniques with behavioral assessments, our study aims to elucidate the intricate interplay between NTS signaling, fast neurotransmitters, and specific circuit activations within the VTA. These findings contribute to our understanding of reward processing and hold potential implications for developing novel addiction treatment strategies.

My research project aims to combat inflammation and fibrosis caused by biomaterials and implants by developing a deeper understanding of the effect of CID on macrophage phenotype. Non-degradable biomaterials can provide long-term stability in the body but can elicit a foreign body response, such as inflammation. The project involves engineered M1 cells which were created and published by the Giachelli and Scatena Lab within the Department of Bioengineering at UW. We have two groups of mice: one control group that has been injected with engineered TLR4 (Toll-Like Receptor 4) cells but not given the CID (Chemically Induced Dimerizers) drug, and another group that has been injected with both the engineered TLR4 cells and given the CID drug. Previous in vitro studies have demonstrated that the engineered TLR4 cells activate the proinflammatory M1 macrophage phenotype when treated with CID. Activation of the proinflammatory M1 phenotype is expected to result in alteration of the healing process, including altering the collagen quantity and structure. At this stage in the project, we have tissue samples from both these groups, and I am currently analyzing these samples using both H&E staining and Picrosirius Red staining thus allowing me to measure healing parameters, like density of collagen. At the same time as data collection, I am using ImageJ to obtain measurements and expect to see the group with CID have a denser collagen structure around the material than the group without CID since CID causes activation of the M1 phenotype. Conducting the analysis on these tissue samples will help address the effect of CID on healing parameters, such as inflammation, and help us develop a better understanding of the roles that M1 and M2 phenotypes play in the healing process.