Figure 1: This is an image of the social behavior plate with the Danio rerio that were exposed to the RLX-33 antagonist. There are clear combs between each pair of fish. The plate was placed in a behavior box where the footage was also recorded.
The goal of this class is to guide us through an independent research project of our choosing. Beginning during the summer, and continuing up until our STEM Fair in February, we started with brainstorming and narrowing down our project idea, then conducting background research and writing a grant proposal, and finally conducting research and writing our thesis. Throughout the course, we practiced a lot of scientific reading, writing, and presentation skills. In terms of our project, we are able to explore any topic of our choosing falling under the category of science, engineering, and/or math. Although this leads to everyone working on unique projects that fall on a wide range, there is still lots of collaboration not only through class work that requires group work, but there are also many oppurtunities for us to get to learn about each others' projects and provide feedback.
Autism Spectrum Disorder is a neurodevelopmental disorder that affects millions of people worldwide. It affects intellectual and social behaviors, often through adulthood. My project specifically focused on the social impairment aspect, observed through zebrafish (my model organism) with a mutation in the human ASD risk gene, ARID1B. The mutated zebrafish had decreased sociability and an increase in the hormone Relaxin-3. So, the goal of my project was to check if their was an association between the increased expression of this hormone and the reduced interactions between the zebrafish. When the zebrafish were exposed to an initial 15µM concentration of the drug that reduced the hormone, it was observed that the zebrafish with the drug had a lower average number of initiated interactions compared to the zebrafish without the drug (p-value: 0.8). It is predicted that this was likely due to an overpowering concentration of RLX-33, so I am planning on testing out various lower drug concentrations in the near future.
Autism Spectrum Disorder (ASD) is a prevalent neurodevelopmental disorder that affects the social and intellectual behavior of many people. Although there are select behavioral therapies and medications currently recommended to alleviate symptoms, all have varying degrees of success and drawbacks. Present research on the biological factors of ASD has led to the discovery of ASD risk genes. A mutation in the Danio rerio ortholog of risk gene, ARID1B, resulted in decreased sociability and increased expression of the Relaxin-3 hormone. This project aimed to identify if the suppression of this hormone would ameliorate sociability. It was predicted that ARID1B mutant Danio rerio administered with the Relaxin-3 antagonist, RLX-33, would have an increase in the number of interactions initiated with their neighboring fish. The hypothesis was tested by immersing ARID1B mutant Danio rerio into the drug solution and then running a social behavior assay. As this was a new drug screening method in Danio rerio, the first goal was to optimize the procedure to maximally gather accurate data on the drug's effect. Determining the ideal RLX-33 concentration was important, as the understanding of small molecule drug concentrations in Danio rerio is not well-established. The initial testing of a 15µM concentration led to the death of half the fish in the plate during the assay. A PCR was later performed to distinguish the tendencies of Danio rerio that had the target gene mutation. The developed analysis pipeline will combine video footage of Danio rerio with PCR results to assess clips from the wells.
Research Question:
What are the effects of the Relaxin-3 antagonist, RLX-33, intake on the sociability deficit of ARID1B mutant Danio rerio?
Research Hypothesis:
It is hypothesized that if the Relaxin-3 antagonist, RLX-33, is administered to ARID1B mutant Danio rerio, then they will have improved sociability in terms of increased initiated interactions.
Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder that affects the learning and social behaviors of millions of people around the world. Their delayed social maturation can make it difficult for them to be integrated members of society during their time in education, in their workplace, and many other public settings. Unfortunately, there are no clear methods of treatment currently that can cure ASD, and many of the medications approved for this disorder primarily target one specific symptom, while causing many other negative drawbacks.
ARID1B
As research regarding the genetic basis of neurological disorders has been increasing, scientists have been using this information to improve early detection and treatment methods. Particularly, dozens of risk genes have been identified to significantly lead to more cases of ASD when a mutation occurs. However, research is ongoing about how the mutations exactly cause ASD symptoms. One of the high-risk genes for ASD is the AT-rich interactive domain protein 1B (ARID1B) encoding gene. Previous studies have identified that mutations in this genome sequence often lead to haploinsufficiency of the protein. The primary function of the ARID1B protein is to regulate gene expression through chromatin remodeling, which refers to the adjustment of how tightly the DNA is wrapped, and hence, how much it is transcribed (ARID1B Gene, n.d.). This gene has been linked with many different intellectual disabilities, and one possible theory is that the ARID1B protein regulates neuron formation through chromatin remodeling in the brain (Sim et al., 2015). In a previous study, an ASD risk gene was applied to Danio rerio through a protein truncation mutation of an ortholog gene of ARID1B in the fish (Capps et al., 2024). This resulted in abnormal amounts of activity, particularly during the day, as well as deficits in sociability amongst the other fish. These are both common indicators of ASD in Danio rerio behavior.
Relaxin-3
Amongst the Danio rerio that were given the ARID1B gene mutant, there was a significant increase in the amount of the Relaxin-3 and Urocortin-3 neuropeptides. Neuropeptides affect neuron activity and regulate the release of neurotransmitters. Relaxin-3 signaling in particular is associated with many different neurological processes relating to arousal, memory, mood, and anxiety. A previous study performed with mice identified that the mice that went through social interaction testing after being injected with the hormone Relaxin-3 performed significantly lower social approaches to other mice, as well as increased passive social contact. RLX-33 is an antagonist that can block this hormone from being expressed in the brain.
Danio rerio breeding
The first step of this project was to cross the ARID1B mutant zebrafish that were already being grown in the UMMS fish facilities. This was done by placing one male and female zebrafish on separate sides of a shallow tank divided by a plastic divider. To accurately time the breeding, the plastic wall is kept between the two fish until the desired breeding time, which is when the divider is removed and artificial plants are added to the tank. The shallow water and plastic plants mimicked the natural breeding grounds of the fish. Two pairs of fish were crossed for this experiment, and this resulted in approximately 400 total embryos across the two clutches. This procedure was performed in order to create the experimental group of zebrafish with the ARID1B mutant.
PCR
Quantitative PCR was run on DNA samples from eight fish from each clutch to identify if the mutation had successfully passed on to the offspring embryos. First, the embryos were hotshot with NaOH to extract the genetic material from the fish. This required neutralization with Tris for the sodium hydroxide. Then, TAQ, primers, and Milli-Q water is added to the DNA before it is amplified while in the PCR machine.
Immersion Exposure
The antagonist RLX-33 was administered to the zebrafish in this experiment through their water. Zebrafish can ingest substances through their mouths, gills, and skin. The solution with the antagonist consisted of blue water, DMSO, and RLX-33. As the walls of the social behavior plate were non-permeable, equal amounts of the antagonist solution were added to each chamber, and then one zebrafish was put into each chamber using a serological pipette. This method of drug exposure was used as a simple yet safe and effective way to administer the antagonist into the zebrafish.
Behavioral Assays
Behavioral assays were performed on the Danio rerio to identify and compare the sociability of the fish in the experimental and control groups. After the social behavior plate with the zebrafish is set up in the behavior box using an acrylic stand and putty to keep it in place, the recognition settings are set up on the computer the system is connected to in order to accurately identify the number of interactions between the fish. This is done by measuring how many times the fish approach the clear wall of the plate where there is a fish on the other side. However, the fish are initially separated by opaque walls to measure their baseline behaviors.
Figure 1: This is an image of the social behavior plate with the Danio rerio that were exposed to the RLX-33 antagonist. There are clear combs between each pair of fish. The plate was placed in a behavior box where the footage was also recorded.
Figure 2: This is an image of the scanned PCR gel for the Danio rerio in the plate with the drug. The upper band is the normal wild type gene while the lower band is the mutant gene. Additionally, row 1 is in every other column.
Figure 3: Scatter plot of the initiated interactions for plate 1 (drugged plate).
Figure 4: Scatter plot of the initiated interactions for plate 2 (control group).
The initial PCR displayed that xx% of the selected fish from the two clutches successfully inherited the ARID1B mutant. Furthermore, the LabVIEW software recorded an average of xxx interactions initiated by the RLX-33 administered ARID1B mutant Danio rerio, while the control group without the RLX-33 exposure averaged xxx interactions. The experimental group also averaged xxx meters of bouts of movement, and xxx seconds of still time. On the other hand, the control group averaged xxx meters of bouts of movement, and xxx seconds of still time. Behavioral Tests The fish were placed in social behavior plates and inside the behavior box to record their activity and exchanges with the fish in other chambers. Initiated Interactions The behavior box quantified the sociability of the fish by measuring the amount of times the fish approached one of the clear, connecting walls of its chambers.
The primary hypothesis being tested was that the ARID1B mutant Danio rerio exposed to the Relaxin-3 antagonist, RLX-33, would have ameliorated sociability. Their amiable behaviors was measured through the number of interactions they initiated with their neighboring fish approaching the clear wall(s) of their chamber. The mean number of initiated interactions of the drug-treated ARID1B mutant Danio rerio during the 90 second period was a. On the other hand, the mean number of initiated interactions for the control group with the ARID1B mutant Danio rerio was b. The mean amount for the experimental group was significantly higher than the mean amount of the control group with a p-value of __. This aligned with the previous understanding that the Relaxin-3 hormone affects sociability of organisms. However, the effect of the hormone suppression was not as drastic as expected. As this was a novel method of drug scanning using behavioral assays, there were many obstacles that created a need for immediate adjustments to the procedure, as well as calculated inferences that were made in order to account for precise, unknown specifics. One of the first challenges that was encountered was manufacturing errors of the social behavior plates. The plates were 3D printed through an outsourced company, but the thickness of the walls were uneven which prevented the clear combs from fitting as they were originally meant to. In order to combat this, we broke apart each of the clear combs into individual dividers and then forced them into the chambers. This warped some of the walls of the social behavior plate, so this likely led to differences in the amount of space each fish had. Additionally, as small molecule drug testing in Danio rerio is not well known, we had to use information known about the administration of RLX-33 on mice, as well as information about its half maximal inhibitory concentration to make an educated inference about the concentration of the drug. However, due to material and time constraints, we were only able to test one concentration. The fist concentration we used, 15 µM, led to the death of over half the Danio rerio in the plate. Therefore, in the future, is has been planned to test out various different concentrations on smaller populations to identify the ideal amount. Through these results, it has supported the connection between Relaxin-3 and sociability. Therefore, more research about this hormone and its inhibitor can be conducted to potentially integrate this knowledge to combat decreased sociability in ASD and other neurodevelopmental disorders.
The objective of this project was to identify if the inhibition of the Relaxin-3 hormone through the antagonist, RLX-33, would ameliorate the decreased sociability in ARID1B mutant Danio rerio. Danio rerio were exposed to the drug through immersion in a solution with 15 µM and 0.15% DMSO and blue water. Then, they were put through a social behavior assay which analyzed how often they approached the clear wall connecting them to a neighboring fish. Before the videos were analyzed, a PCR was run on the Danio rerio DNA samples to identify the heterozygous mutants. The behavior of these mutant fish were focused on during the analysis of the videos. The analysis revealed that xxx. This information can be used to develop a stronger understanding about how intellectual traits, such as sociability, are affected by neurological processes. Additionally, these findings can help open up avenues to treatment of specific symptoms, such as decreased sociability, in people with ASD and other neurodevelopmental disorders.