In seasonally breeding vertebrates, hormones coordinate changes in nervous system structure and function to facilitate reproductive readiness and success. Steroid hormones often exert their effects indirectly via regulation of neuromodulators, which in turn can coordinate the modulation of sensory input with appropriate motor output. Female plainfin midshipman fish (Porichthys notatus) undergo increased peripheral auditory sensitivity in time for the summer breeding season, improving their ability to detect mates, which is regulated by steroid hormones. Reproductive females also show differences in catecholaminergic innervation of auditory circuitry compared to winter, non-reproductive females as measured by tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholaminergic synthesis. Importantly, catecholaminergic input to the inner ear from a dopaminergic-specific forebrain nucleus is decreased in the summer and dopamine inhibits the sensitivity of the inner ear, suggesting that gonadal steroids may alter auditory sensitivity by regulating dopamine innervation. In this study, we gonadectomized non-reproductive females, implanted them with estradiol (E2) or testosterone (T), and measured TH immunoreactive (-ir) fibers in auditory nuclei where catecholaminergic innervation was previously shown to be seasonally plastic. We found that treatment with T, but not E2, reduced TH-ir innervation in the auditory hindbrain. T-treatment also reduced TH-ir fiber density in the forebrain dopaminergic cell group that projects to the inner ear, and likely to the auditory hindbrain. Higher T plasma in the treatment group was correlated with reduced-ir TH terminals in the inner ear. These T-treatment induced changes in TH-ir fibers mimic the seasonal downregulation of dopamine in the midshipman inner ear and provide evidence that steroid hormone regulation of peripheral auditory sensitivity is mediated by dopamine.

Recreating the signaling profile a chemical synapse to analyze serotonin receptor activation is a challenge. This is due in part to the kinetics of the synapse, where neurotransmitters are rapidly released and quickly cleared by active reuptake machinery. One strategy to produce a rapid rise in a bio-orthogonally controlled signal is via photocaged compounds. In this work, a complementary pair of BODIPY photoremovable protecting groups was conjugated to a 5HT2C subtype selective agonist, WAY-161503, and antagonist, N-desmethylclozapine, to generate “caged” versions of these drugs. These conjugates can release their bioactive drug upon stimulation with green light (agonist) or red light (antagonist). We report on the synthesis, characterization, and bioactivity testing of the conjugates against the 5HT2C receptor. We then characterize the kinetics of photolysis quantitatively using HPLC and qualitatively in cell culture conditions stimulating live cells. The compounds are shown to be stable under dark conditions for 48 hours at room temperature, yet photolyze readily when irradiated with visible light. In live cells expressing the 5HT2C receptor, precise spatiotemporal control of the degree and length of calcium signaling is demonstrated. By loading both compounds in tandem and leveraging spectral multiplexing as a non-invasive method to control local small molecule drug availability, we can reproducibly initiate and suppress intracellular calcium flux on a timescale not possible by traditional methods of drug dosing. These tools enable a greater spatiotemporal control of 5HT2C modulation and will allow for more detailed studies of the receptors signaling, interactions with other proteins, and native physiology.

A novel soft actuator is designed, fabricated, and optimized from liquid marbles encased via interfacial polymerization for added mechanical strength and robustness. They are encased in bimorph-type soft actuators where one side of the actuator has a dramatically different Young’s modulus than the other, leading to directional actuation which is successfully demonstrated in multistep walking soft robots. The soft actuators were also shown to successfully activate the mechanosensitive Piezo protein in a transfected human cell line, 293T. Overall, the liquid marble powered soft actuators described here represent a new soft actuation methodology and a novel tool for mechanobiological studies.

Dopaminergic pathways control highly consequential aspects of physiology and behavior. One of the most therapeutically important and best-studied receptors in these pathways is dopamine receptor D2 (DRD2). Unfortunately, DRD2 is challenging to study with traditional molecular biological techniques, and most drugs designed to target DRD2 are ligands for many other receptors. Here, we developed probes able to both covalently bind to DRD2 using photoaffinity labeling as well as provide a chemical handle for detection or affinity purification. These probes behaved like good DRD2 agonists in traditional biochemical assays and were able to perform in chemical biological assays of cell and receptor labeling. Rat whole brain labeling and affinity enrichment using the probe permitted proteomic analysis of the probes’ interacting proteins. Bioinformatic study of the hits revealed that the probes bound non-canonically targeted proteins in the Parkinson’s disease network as well as the retrograde endocannabinoid signaling, neuronal nitric oxide synthase, muscarinic acetylcholine receptor M1, GABA receptor, and dopamine receptor D1 (DRD1) signaling networks. Follow-up analysis may yield insights into how this pathway relates specifically to Parkinson’s disease symptoms or provide new targets for treatments. This work reinforces the notion that the combination of chemical biology and omics-based approaches provide a broad picture of a molecule’s “interactome,” and may also give insight into the pleiotropy of effects observed for a drug, or perhaps indicate new applications.