Hello, my name is student Dr. Belucci, and I would like to discuss the project I have been working on at Toro University, California. The goal of our project was to better understand the role 5-HT2A and 2C receptors played during differentiation of neural crest-derived tissue and ultimately the effect they have on embryological development. I'd like to start off with a brief introduction of serotonergic hallucinogens. These drugs induce neuroplasticity and increase dendritic spine density through release of cell-signaling molecules and gene induction. This has been studied in many areas of the brain, but of most relevance are their effects on the mood-regulating regions of the brain. These changes in neuroplasticity and subsequent antidepressant activity of these drugs are likely due to their activation of 5-HT2A and 2C receptors. 2C receptors also inhibit the release of dopamine, and up-regulation of 2A and 2C receptors has been indicated in depressive and anxiety disorders. Interestingly, despite differing targets and mechanisms of actions, both antidepressant medications and serotonergic hallucinogens often down-regulate 2A and 2C receptors. This has made these hallucinogens an area of interest in treatment of depressive disorders. Unfortunately, our understanding of their effects and mechanisms are still somewhat limited. For example, in mothers taking an SSRI, the medication crosses the placenta to the developing fetus. Similarly, serotonergic hallucinogens are also taken up by the fetus. 2A and 2C receptors play a significant role during a neurological development, and of specific interest to our experiment is their effects on the development and differentiation of neural crest cells. These cells give rise to many structures in humans, including the dorsal root ganglia and the autonomic nervous system, pigment cells, and craniofacial and cardiac structures. So altering this pathway early during fetal development could result in significant morphogenic changes. Our project sought to clarify this interaction by studying how the signaling activity of serotonin receptors 5HT2A and 2C modulate morphogenesis of neural crest cell-derived tissue, specifically in Xenopus laevis embryos. Because neural development and pathways are highly conserved between Xenopus and humans, they serve as an excellent model organism for our purposes. For large embryos, quick external development and well-defined embryological stages allow for easier manipulation and better imaging. We began by mating Xenopus laevis and collecting embryos the following day. The embryos were then de-jellied, washed, and allowed to grow until the early tailboot stage. The embryos were then transferred to one of three dishes, one containing 5-micromolar 5HT2A agonist, namely 25CNBOH, one containing 5-micromolar 5HD2C antagonist, namely SB242084, or the control dish which only contained MMR solution. They were then allowed to develop at room temperature until the late tailboot stage. At this stage, the embryos were anesthetized in Trican and fixed using paraformaldehyde for one to two hours. Following this fixation process, the embryos were rinsed in PBS and stored for imaging. Imaging was done using a Nikon SMZ500 dissecting microscope. The embryos were transferred to a clay-lined Petri dish filled with PBS solution. They were then oriented to image them dorsally and laterally. Their heads were then transected from their bodies to allow for frontal images of the head. Image J was used to measure the images. Images captured the size as well as the shape of the embryo. The heads, eyes, chord, fissure, and lateral band of pigment cells is what we measured. These structures are derived from neural crest cells in Xenopus laevis. Measurement values were for area, perimeter, circularity, and aspect ratio. Area and perimeter were used to measure changes in size. Aspect ratio and circularity were used to measure changes in shape. A two-sample unequal variance t-test was done for statistical analysis, and our sample size were as follows. 10 for the 2A agonist group, 19 for the 2C antagonist group, and 14 for the control group. To start off our findings, I would like to discuss cranial morphogenesis. The 2A agonist embryos had an increase in the roundness of the head, while the 2C antagonist embryos had smaller and more elongated head when we compared them to the control. With eye morphogenesis, the 2A agonist embryo eyes and chord fissures were larger, while the 2C antagonist embryo eyes and chord fissures were smaller and malformed when compared to the control. With the lateral pigment cells, both the 2A agonist and 2C antagonist embryos had a decrease in the size of the lateral band when we compared to the control. And all of these findings were statistically significant. So our initial question was, do 5HT2A and 5HT2C receptors alter the embryological morphogenesis of neural crest-derived tissues? And if they do, might these changes apply to human fetuses and to serotonin-modulating medications? What the changes seen in the embryos administered the 2A agonist or 2C antagonist, when we compared them to the control, it follows that their respective receptors are likely responsible for these morphological differences. If the embryo is exposed to chemicals that alter their physiologic activity of these receptors long enough and at a high enough of a concentration, we expect there to be permanent changes to structures derived from these neural crest cells. Considering the permeability of serotonergic hallucinogens and serotonin-modulating agents, physicians should be informed of the risk these drugs hold for pregnant people and the need for further research surrounding this topic. Thank you.

5-HT2A receptors

neural crest

Xenopus laevis

embryological development

serotonergic agents