2025 Theses Doctoral

Development and Characterization of Fluorescent Probes for Understanding Monoamine Neurotransmitter Pathways in the Brain

Mendieta, Adriana

The topics and projects outlined in this doctoral thesis review the development of chemical tools for studying specific proteins in relation to serotonergic system expressed in native brain tissue to gain a broader understanding of serotonin neurotransmission in the brain. Chemical-based probes without need of invasive genetic manipulation to function assist in visualization and circuit mapping of membrane bound proteins associated with serotonin in native tissue.

Chapter 1 introduces key proteins crucial for serotonin movement within the brain, how antidepressant medications impact serotonin levels, why psychedelics are hypothesized to be fast-acting antidepressants, and why developing new fluorescent imaging agents for pinpointing localization of the receptor target associated with psychedelics within the prefrontal cortex is crucial for elucidating their mechanism of action. The new series of probes created from an extensively studied and established small molecule ligand and tethered to commercial fluorescent dyes show promising results in cell culture and brain tissue slices.

Chapter 2 expands on the background of the serotonin transporter, what key function does it serve in the brain, why we need to introduce the novel fluorescent probe SERTLight that selectively labels cells expressing this transporter and discusses the establishment of new cell-based assay protocols to characterize SERTLight at a subset of central and peripheral monoamine transporters, classified as Uptake 2. SERTlight demonstrated to have no inhibitory or substrate activity at Uptake 2, reinforcing the claim of SERTLight’s selectivity for the serotonin transporter.

Chapter 3 provides background regarding the previously mentioned Uptake 2 transporters that assist in clearing chemical messengers like endogenous serotonin in the extracellular space. From the SERTLight work, cell assays showed the existence of Fluorpts270, a variation of SERTLight with substrate activity at both serotonin and the Uptake 2 transporters. Fluoropts270 is an attractive fluorescent reporter capable of tracing the degree of serotonin uptake within both sets of transporters.

Chapter 4 introduces the natural product ibogaine and several novel derivatives shown via confocal microscopy to inhibit uptake mediated by serotonin transporter uptake, vesicular monoamine transporter 2 uptake, or both. Compounds capable of both at near equal potencies inspired the idea of a new class of compounds termed synaptic reuptake inhibitors, or SynRI. Chapter 4 will discuss the exploratory work of investigating the pharmacological properties several iboga alkaloids in Uptake 2 transporters.