A product we consume daily, such as honey, may contain heavy metals like copper (Cu). Copper can get into our environment and food from urban sources such as tires, roofing materials, and generators. We therefore expect food products from more urbanized areas to show a higher concentration of copper. To test this hypothesis we evaluated copper levels in raw honey from different regions with different levels of urbanization. The honeys we tested were divided into three different categories according to their source: rural, suburban, and urban. The samples were digested by reflux with HNO₃ and H₂O₂ then it was diluted and filtered in preparation for analysis by Atomic Absorption Spectroscopy. The results suggest that the honey with the highest concentration of Cu was from an urban area, and the honey from a rural setting had the least amount of Cu. A one-way ANOVA analysis confirmed a statistically significant difference in the copper levels in raw honey from the regions (p < 0.01). The concentration of Cu in the samples of honey we analyzed ranged from 0.2 ug/g to 0.4 ug/g which is well within the recommended upper limit of 300 ug/g. Since bees collect pollen and honey from plants within roughly one mile of their hive, evaluation of the levels of copper and other metals in honey can provide a snapshot of the background levels of exposure in that area.

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.