Category: College of Humanities and Social Science

Spectroscopic characterization of newly designed fluorescent probes for biomedical research

Author(s): Amira Anwar

Mentor(s): Ozlem Dilek, Chemistry & Biochemistry

Abstract

The development of fluorescent probes is crucial for advancing cellular imaging and disease diagnostics, particularly in detecting oxidative stress, a key driver of cancer, neurodegenerative diseases, and fibrosis. A hallmark of oxidative stress is the carbonylation of biomolecules, which occurs when reactive oxygen species (ROS) modify proteins and other biomolecules, leading to cellular dysfunction. In this project, we designed and synthesized hydrazine-based small-molecule fluorophores to selectively target biological aldehydes, particularly those generated by lysyl oxidases (LOXs) during collagen oxidation. These aldehydes serve as important biomarkers of fibrosis and metastatic progression. The fluorophores react with aldehydes to form hydrazones, resulting in a fluorescence turn-on response that enables real-time monitoring of oxidative stress in cellular systems. To characterize probe performance, we analyzed their photophysical properties using UV-visible and fluorescence spectroscopy. Additionally, reaction kinetics were evaluated via UV-visible spectrophotometry, and structural validation was conducted using NMR and mass spectrometry. By refining probe design for enhanced fluorescence sensitivity and specificity, this work contributes to the development of next-generation imaging tools for studying disease mechanisms and advancing biomedical diagnostics.

Audio Transcript

Hi everyone, and thank you for checking out my presentation. I’ll be sharing my research on the spectroscopic characterization of newly designed fluorescent probes for biomedical research. These probes are designed to detect biologically relevant aldehydes—aldehydes are highly reactive molecules that can build up in the body during oxidative stress. This kind of cellular stress is involved in many major health conditions like cancer, fibrosis, and neurodegenerative diseases. By creating tools to visualize these aldehydes in real time, we hope to provide a better way to study early-stage disease and potentially improve how we diagnose and/or monitor these conditions. So, why is this important? Our bodies naturally produce molecules called reactive oxygen species, or ROS. But when there’s too much ROS and not enough defense, it leads to oxidative stress. This damages important parts of our cells—proteins, DNA, lipids, and nucleic acids. One of the results of this damage is something called carbonylation, an irreversible modification to proteins that disrupt its normal function which serves as a biomarker for disease progression. By detecting this early and accurately could give us new ways to track disease or catch it early—especially in conditions like cancer or metabolic disorders. Right here is an image of a normal cell being attacked by free radicals, elevated levels of ROS leads to oxidative stress. The role of LOX. A group of enzymes called lysyl oxidases, or LOX are important in building collagen, the main protein in connective tissues. But during that process, they produce aldehydes.These aldehydes, especially when overproduced, are closely tied to tissue damage and disease. So if we can track them, we can better understand what’s going on in the body during early disease stages.So this ia relaly good image on how LOX cross linking collagen occurs and potential way to image LOX. To tackle this, I designed a small molecule—called fluorescent probe—that glows when it detects aldehydes. The idea is simple: no aldehyde, no glow. But when an aldehyde is present, the probe reacts and lights up.This happens via click chem mechanism Shown right here. This kind of “turn-on” fluorescence lets us track where and when aldehydes are forming, even in real-time, which is incredibly useful for research and diagnostics. Here’s a detailed view of how it works: We start with an amine which converts into a hydrazine molecule—Molecule 5. This hydrazine reacts specifically with aldehydes. Once it does, the product becomes a fluorescent hydrazone molecule. We tested this with two model aldehydes: formaldehyde and butyraldehyde. And we saw that when the reaction happens, there’s a clear increase in fluorescence.To study the probe’s behavior, we used tools like UV-Vis and fluorescence spectroscopy. These techniques let us measure how much light the molecule absorbs and emits. Our data showed that after reacting with butyraldehyde, there was a strong increase in fluorescence. That confirmed the probe was working just as we hoped. We also wanted to know how fast this reaction happens and in what environments it works best.In our kinetic studies, we found that the probe reacted more quickly with butyraldehyde than with formaldehyde, and it performed better in methanol than in pbs which is similar to human body ph 7. Even better, the reaction happens in seconds, which is perfect for real-time tracking. Here’s a visual. In these three vials, you can see how the probe behaves: The first vial just has the probe in methanol—no fluorescence. The second has methanol, butyraldehyde and the probe—strong fluorescence. The third has methanol, formaldehyde and the probe—some fluorescence, but not as much.So, in summary, we’ve developed a fluorescent probe that’s small, fast-reacting, and selective for aldehydes. It gives a clear signal when aldehydes are present, which opens up exciting possibilities for detecting biologically relevant aldehydes, with an emphasis on those generated by lysyl oxidases (LOX). This tool could help researchers track disease progression or even assist in diagnostics in the future. Our next steps include:Testing the probe in live cells to see how it performs in biological systems. Making more versions of the probe, including ones that work in the near-infrared range for deeper imaging in tissues. And eventually, applying it in 3D printed tissue models or even live animal studies.Thank you so much for watching!

College of Humanities and Social Science OSCAR

Assessing Soil Organic Matter In a Legacy Wetland Mesocosm after the Colonization of Invasive Species

Author(s): Trinity Lavenhouse

Mentor(s): Changwoo Ahn, Environmental Science and Policy Department

Abstract

This study was conducted in Dr. Ahn’s wetland mesocosm on West Campus at George Mason University. Inside there are 40 wetland mesocosms that were created in 2012. The mesocosms were initially planted with 0 to 4 different native wetland species. Since COVID-19, maintenance halted which allowed the colonization of invasive species for the past three to five years. The goal of this study was to determine the amount of soil organic matter in each mesocosm after the colonization of invasive species. The hypothesis was that because there were more plants than there were previously, there will be a higher amount of soil organic matter in these mesocosms. The soil organic matter percentage was processed using the loss on ignition method. It was found that mesocosms that had sparsely spread, or no plants had the lowest soil organic matter while overgrown and dense mesocosms had the highest soil organic matter. After about 10 years, soil organic matter seemed to increase from a range of 2.36% to 2.59% to now a range of 4.15% to 6.21%. In the future a soil bulk density will be processed to analyze how soil porosity or compaction correlates with the soil organic matter found within the mesocosms. Also, plant identification will be conducted for all 40 mesocosms.

Audio Transcript

Hello, my name is Trinity Lavenhouse, and I am presenting my research on assessing soil organic matter in a legacy wetland mesocosm after the colonization of invasive species. My mentor is Dr. Changwoo Ahn.
So, to start off I am going to explain the importance of measuring soil organic matter! Soil organic matter is made up of decomposed plant and animal material. They contain quite a lot of organic compounds and are full of nutrients that help plants grow. So in general, it is important to measure the amount of soil organic matter that is present because it can indicate how healthy and fertile the soil is for plants to grow.
This study was conducted in Dr. Ahn’s wetland mesocosm on West campus in George Mason University. Inside there are 40 wetland mesocosms that were created in 2012. The mesocosms were initially planted with 0 to 4 different native wetland species. This picture is from 2014 after 2 growing seasons. Since COVID-19, maintenance halted. Unwanted plants weren’t removed and the water levels weren’t monitored like they use to be. This allowed the colonization of invasive species for the past three to five years. So, the goal of this study was to determine the amount of soil organic matter in each mesocosm after the colonization of invasive species. The hypothesis was that since there were more plants than there were previously, there will be a higher amount of soil organic matter in these mesocosms.
To take samples the mesocosms were split into 5 sections and one sample was taken from each section within the top 20 centimeters where organic matter is the most present, because soil organic matter decreases as you go further down. This was repeated for all 40 mesocosms. The soil organic matter percentage was processed using the loss on ignition method. To carry this out each sample was dried at 105°C for two days. After they were completely dry, they stayed in a desiccator to cool and were then crushed in with a mortar and pestle and sieved through a 2mm filter to get everything that is too big to go into the crucibles. The fine dirt was then placed in crucibles and fired at 360 Celsius for two hours and they were placed back into the desiccator to cool. Soil organic matter percentage is the percentage change of mass.
The results were all collected and all five of the samples were compiled and averaged represent the one mesocosm. It was found that mesocosms that had sparsely spread, or no plants had the lowest soil organic matter while overgrown and dense mesocosms had the highest soil organic matter. The standard error bars are large in mesocosms that had more plants or organic matter in a section than other sections. The mesocosms were then grouped by the initial number of species planted. There seems to be no significant difference across the board and the reason for that will be analyzed further.
In a previous study conducted by another person in this lab named Mary Means, they measured the total carbon content of all of the 34 planted mesocosms in 2014. Total carbon content is a is about 58% of soil organic matter so that conversion was used to estimate what the soil organic matter content was back in 2014. About ten years until now, later soil organic matter seemed to increase from a range of 2.36% to 2.59% to now a range of 4.15% to 6.21%. In the future a soil bulk density will be processed to analyze how soil porosity or compaction correlates with the soil organic matter found within the mesocosms. Also, plant identification will be conducted for all 40 mesocosms as when these samples were initially taken most of the plants were dead because it was the end of the winter.
Thank you for listening!

College of Humanities and Social Science OSCAR

Evaluating Perceptions of Basic Psychological Needs in Full-Time Employees

Author(s): Jessica Luther

Mentor(s): Lauren Kuykendall, Psychology

Abstract

The climate of the current workplace has changed drastically within the past few years, as a result of many jobs moving to remote work. Many employees have had to learn how to adjust to dealing with the physical separation of leadership, as well as balance their own basic psychological needs in a new environment. These three needs, autonomy, competence, and relatedness, are important workplace predictors that still need further exploration in the remote workplace. This study samples from both remote and in-person workers, providing a clear comparison of how the change in workplace setting impacts an employee’s perception of leader-member quality, as well as their basic psychological needs. We hypothesized that LMX would have a significant and positive impact on each basic psychological need. We also hypothesized that remote work will moderate the relationship between LMX and each basic psychological need, such that the positive relationship will be stronger for non-remote workers than remote workers. Participants consisted of 241 full-time employees, 123 in-person workers and 118 remote workers. Results show that while the correlation between LMX and each basic psychological need were significant, they were not statistically different between work locations. This has implications for how the basic psychological needs can be fulfilled in the remote environment, challenging the assumption that the in-person environment is better at fostering better leader relationships and significance of the impact on the employee.

Audio Transcript

Hi everyone! My name is Jessica Luther and this is my project, Evaluating Basic Psychological Needs in Full-Time Employees. This study was done with the help of Dr. Kuykendall, and represents my research conducted for the third semester of the Honors in Psychology program. Since COVID, there’s been more flexibility in allowing employees to have part-time and full-time positions working remotely. Prior research shows that perception of leader quality is a predictor for employee well-being which has been studied in both the in-person and remote environments. The perception of someone’s basic psychological needs has also been seen to change the remote environment, a result of whether it be not seeing people, or whether it be feelings of social isolation. Basic psychological need perception has also changed. Connecting these three topics, since COVID we have seen a lot of people moving to remote positions, and a lot of the recent research in remote work kind of reflects how leader quality or leadership styles are impacting employees, specifically, the outcomes such as autonomy, motivation, a lot of stress-related outcomes as well. So, for this study to provide a direct comparison between in-person and remote employees we looked at how leader member exchange, which is noted as LMX, or also known as the perception of leader quality, is moderated by remote work and how remote work changes the relationship that someone might have with their leader or the perception of the quality they have with their leader and how that impacts their three basic psychological needs, which are autonomy, competence, and relatedness. My hypotheses are split into two parts, you have hypotheses 1a through 1c. This deals with direct associations between LMX and autonomy, LMX and competence, and LMX and relatedness. Prior research shows LMX is positively related to autonym, competence and relatedness, so we assume the same thing for this study. Hypotheses 2a, 2b, and 2c are where my contribution to the research comes in. It suggests that remote work moderates this relationship so remote work will moderate the relationship between LMX and each basic psychological need, such that the positive relationship will be stronger for no-remote workers than remote workers. Basically, insinuating that we will see that autonomy, competence and relatedness are both significant and stronger for in-person employees rather than remote employees. So, my participants were 241 full-time employees, 51% worked in-person, 49% worked remotely. We did not include any hybrid workers or anyone who worked part-time just to have a more balanced sample of who we were sampling from so full-time in this case was defined as working 35 plus hours per week. 42.3% were male, 55.2% were female, and 2.5% identified as non-binary. They were excluded if they worked part-time, were hybrid workers, or resided somewhere other than the U.S.. So, the methodology for this was that they were recruited on Prolific, which is an anonymous survey taking website, and the survey itself took place on Qualtrics. The two instruments used were the LMX-7, which measures perceived leader-member quality, or leader-member exchange, and Self Determination Theory 21, or SDT-21 Basic Psychological Needs at Work Scale, which measures each basic psychological need through statements that were tested using a Likert scale. Participants, for their participation, were compensated 2.85%. So, for the results, we ran a correlational analysis between LMX and SDT, separating remote participants from in-person participants, so that we would be able to directly compare their results. We actually found that each component of self-determination theory, autonomy, competence, and relatedness, was significant with LMX in both groups. So Hypotheses 1a-1c were supported, which was expected because we’ve already seen this in prior research, However, Hypotheses 2a-2c were not supported. Even though each component was significant for in-person, through further testing we found that while everything was significant, they were significantly different so the correlations between LMX and SDT of in-person workers was not significantly different than LMX and SDT of remote workers. That suggests that these needs can be filled in both environments. The implications for this show how leadership perceptions might not really change based on environments, and even then, it’s not statistically significant between the two environments themselves, and actually remote work can still be a supporting environment, given that people have supportive leadership roles and supportive leaders that they can count on. One of the studies that I read noted that social isolation was a really really big concern, which could really damage someone’s feelings of relatedness. However, as seen in the table, relatedness was still significant in remote workers using LMX, so having that supportive leadership role really can help fill the need for relatedness. Further research should take into account what hybrid work or part time work might reveal because those are really samples as well. Knowing how different samples perceive different leadership styles is really important to understanding how leaders can support their employees and how leaders can really help fulfill some of these psychological needs. For my acknowledgements, I wanted to say that this couldn’t have been done without OSCAR funding, it was used to help compensate the participants for their participants, and I also wanted to thank Dr. Brielmaier, Dr. Zaccaro, and Dr. Z for all their help in my project as well. Thank you.

College of Humanities and Social Science OSCAR

Predictors of Young Adults’ Physical Activity and Sedentary Behavior

Author(s): Matthew Burdick

Mentor(s): Jerome Short, Psychology

Abstract

Although cardiorespiratory fitness and muscular strength are the strongest predictors of lifespan examined in epidemiologic and experimental research, over 75% of adults in the United States fail to meet national physical activity (PA) guidelines and many live sedentary lifestyles. Identifying predictors of PA and sedentary behavior (SB) may inform public health interventions. This study uses questionnaire and activity monitor data to identify predictors of PA/SB for young adults. Young adults are an underexamined population within the PA/SB literature and research on this topic has yielded inconsistent findings. Participants (n = 216) were about 20 years old, predominantly female (n = 149), and owned a smart watch. The study transpired over 29 days with a pretest and two posttests. We examined 6 predictors: perceived stress, anxiety, depression, somatic pain, meaning in life, and gratitude. Sitting time was significantly negatively correlated with meaning in life, moderate PA, vigorous PA, and exercise satisfaction. Moderate PA and exercise satisfaction were unique predictors of sitting time. Perceived stress and somatic pain were negatively correlated with vigorous PA and steps per week. Sitting was negatively correlated with days of walking. Exercise satisfaction was a unique predictor of vigorous PA and daily steps. We found bidirectional relationship between perceived stress and vigorous PA that was mediated by exercise satisfaction. Our findings indicate that interventions should focus on lowering sitting time and raising exercise satisfaction to promote young adults’ adherence to CDC PA recommendations. The relationship between psychological factors and PA might be largely explained by exercise satisfaction.

Audio Transcript

Hello everyone. So exercise. We all know fundamentally that exercise is probably good for us. It might make us lose some weight. It might help us be more energetic throughout the day. It even might make us live longer. So why is it so hard for us to exercise? So. Research shows that cardio respiratory fitness, and muscular strength are the strongest predictors of lifespan in the research.

So this beats cancer risk. This beats smoking risk. This beats type two diabetes. If you can run fast and hard and you can lift heavy, you will likely live for a long time. Despite this, over 75% of adults in the United States fail to meet the national guidelines. So why is this the case? So our question was, what are the mental and physical factors that predict exercise and sitting behavior, especially in young adults?

And the way we attacked this question was we recruited over 200 Mason students who with a mean age of 20 years old, and we required them to wear smartwatch smart watches throughout a 29 day period. And now throughout that period we analyze their sort of different psychological and physical variables through questionnaire data.

And after that study we were able to run data analysis using SPSS.

So what predictors or did we examine in this study? So we examined sort of two classes of predictors that we hypothesized to be either protective or harmful to physical activity. So protective. We have meaning in life. So the sense that someone feels that they can engage purposefully in their day-to-day lives, sort of the antithesis to nihilism.

And now you have gratitude is gratitude can be a state and a trait so people can feel grateful. All of the time. Or they can have moments where they feel gr grateful, which is more of the trait like gratitude. Um, this questionnaire measures both the state and the trait, but it basically means how grateful are you or like how much gratitude do you have for your current situation despite all the negatives.

So it’s sort of like almost an optimistic worldview. We also have several risk factors, so perceived stress. Anxiety, somatic symptoms and depression. Somatic symptoms meaning bodily pain and aches.

So what do we hypothesize?

First things first, we hypothesize that the risk factors, so remember, that’s your perceived stress, anxiety, depression, and body pain. We predicted that that would be. Um, related to less exercise and more sitting. So for example, someone who is very anxious, we expect them to exercise less and sit more. And this is in line with the research.

Um, uh, alternatively, we think that the protective factors, so those are our median life and gratitude will be related to more exercise and less sitting. Finally, we predict that all these factors to both risk and protective factors will uniquely predict exercise in sitting. So that means that out of all the variables that we assessed in our study, we would think that the, all our protective and risk factors accountant for unique variants in our outcomes.

So what we observe. Our relationship between our predictors and outcomes is primarily attributed to our factors instead of some other confounding third variable. So what do we find? So first we found some pretty interesting information about our samples activity. So again, we have about 200 Mason students over 18 years old.

With a mean age of 20 years old, and we found that GMU students surpass Americans in meeting C to C guidelines. So that means that you can see in our figure here that almost 54% of American, or sorry, 54% of GMU students meet or exceed the national guidelines of activity. So the CD. C recommends that people, especially adults, more specifically.

Participate in at least 150 minutes. So two and a half hours of moderate intensity exercise per week, or the equivalent of vigorous activity. And so we found that in our sample over almost 54% of our students met or exceeded these guidelines. Whereas nationally, only 25% of American adults meet those guidelines.

So we’re almost twice as compliant. Compared to the average American, but this can be somewhat misleading because as you can see on our bottom bar here, the sedentary bar, almost 20% of GE students in our sample had zero minutes, zero minutes of activity per week. And so that was a cause for concern.

We found that meaning in life was negatively related to sitting time. So people who perceive that they have a purposeful engagement in their life, that they can traverse the day-to-day events of their life and feel like it has purpose. Those people tended to sit less throughout this 29 day period.

We also found that people who were physically active, especially within the moderate physical activity category, um, this uniquely predicted sitting time up to 29 days after baseline. So basically this was our big longitudinal finding that people who were. Especially active at day one of our study tended to sit less up to almost a month after baseline.

Additionally, we found that perceived stress and somatic pain were negatively related to vigorous activity and daily steps. So people who reported being more stressed and having more bodily pain, typically exercised less and walked less.

So exploratory analysis revealed some interesting results. So especially before examining the relationship between stress and vigorous activity. As you can recall, before we found that stress was negatively related to physical activity, but uh, exploratory analysis revealed that stress was not a unique predictor of vigorous activity, meaning that.

There’s something else accounting for that variance. And what we found was exercise satisfaction accounted for that variance in a bi-directional, fully mediated model. So what does that mean? It means that perceived stress has no relationship, uh, has at least no unique relationship with vigorous activity.

But when you include exercise satisfaction. It has a mediaing relationship so that, so in our first model here in Model A, we found that people who were stressed reported less exercise satisfaction and exercise satisfaction. Being a strong predictor of vigorous activity would increase vigorous activity.

So basically, people who were stressed had less as facts from exercising, and so were less likely to exercise. Additionally, this was bidirectional. So if you flip it on its head, it is also true. So vigorous activity has no unique predictive relationship with perceived stress, but when you add exercise satisfaction, vigorous activity can, is, can be associated with a lot of exercise satisfaction.

And in doing so since exercise satisfaction is, is, um, associated with less perceived stress. It can vigor activity, could then could lead to less perceived stress. I should state that this is cross-sectional data, so we’re not making a causal claim, but we did find a fully mediated cross-sectional model with these variables, some things to take away with you.

So median life is negatively related to setting time. So. If you can find a way to increase your perception of life being purposeful to you, that things aren’t meaningless, that your actions matter. If you can increase that, you may be able to sit less and improve your physical health. Um, being active, especially moderately active, decreases your risk of sitting for chronically amount, chronic amounts of time.

Uh, stress may reduce exercise satisfaction, which in turn would reduce your, um, vigorous physical activity and vigorous activity may raise exercise satisfaction. And finally, satisfaction is weigh in more vigorous activity. Thank you for listening. Have a good day.

College of Humanities and Social Science College of Science OSCAR

Pharmacological Characterization of Chimeric NMDA Receptors in Xenopus

Author(s): Diborah Gutema

Mentor(s): Theodore Dumas, Department of Psychology, Interdisciplinary Program in Neuroscience

Abstract

NMDA receptors (NMDARs) play a critical role in synaptic plasticity and cognitive function by mediating both ionotropic and non-ionotropic signaling. During postnatal development, NMDA receptors undergo a subunit shift from GluN2B to GluN2A, a transition associated with synaptic maturation and the emergence of mature cognitive function. While previous research has explored the physiological and behavioral consequences of this transition, the specific mechanisms driving these changes remain unclear. In particular, the relative contributions of ionotropic and non-ionotropic signaling have not been fully defined. Ionotropic signaling involves calcium influx through the receptor channel upon ligand binding, while non-ionotropic signaling refers to intracellular signaling cascades initiated by conformational changes in the receptor that occur independently of ion flow. This study aims to dissect these signaling pathways by utilizing chimeric GluN2 subunits engineered to separate ionotropic and non-ionotropic functions. Chimeric constructs are currently being subcloned into Xenopus laevis–compatible vectors for functional expression in oocytes. This work has involved preparation of the pGEMHE-membrane-EGFP backbone through bacterial culture and plasmid isolation via alkaline lysis mini prep, followed by gel electrophoresis and spectrophotometric analysis to assess plasmid integrity and purity. Restriction digests were performed to linearize the plasmid and confirm backbone identity. In parallel, GluN2A, GluN2B, and chimeric constructs have been cultured from glycerol stocks and are currently undergoing PCR amplification with construct-specific primers. Following amplification, the inserts will be digested, purified, and ligated into the vector. Preliminary results confirm successful plasmid preparation, and insert amplification and ligation are ongoing. Once subcloning is complete, the constructs will be injected into Xenopus laevis oocytes, followed by two-electrode voltage clamp (TEVC) recordings to measure receptor responses to varying concentrations of glutamate and glycine, as well as pharmacological modulators of NMDA receptors. Statistical analyses using two-way ANOVA will compare current amplitudes across receptor type (GluN2A, GluN2B, ABc, BAc) and treatment condition (agonist/modulator concentration), with the goal of identifying distinct electrophysiological profiles associated with each subunit composition. This study will characterize how differences in GluN2 subunit composition and intracellular domain identity affect NMDA receptor-mediated signaling in an isolated system.

Audio Transcript

Hello all. I am Diborah Gutema and this is my video presentation for my project, Pharmacological Characterization of Chimeric NMDA Receptors in Xenopus laevis Oocytes.

NMDA receptors are ion channels located on neurons that allow calcium ions to enter the cell when activated by the neurotransmitter glutamate. This calcium signaling, known as ionotropic signaling, is critical for synaptic plasticity, learning, and memory. NMDA receptors can also engage in non-ionotropic signaling, where conformational changes in the receptor trigger internal signaling pathways without ion movement. Each receptor is composed of two GluN1 subunits and two GluN2 subunits. A developmental shift occurs where GluN2B subunits are gradually replaced by GluN2A, a transition essential for synapse maturation. Understanding how these subunits contribute to ion flow and conformational signaling is the focus of our project.

To investigate how different regions of NMDA receptor subunits contribute to signaling, we are working with chimeric GluN2 constructs developed by Dr. Dumas’s lab. These chimeras are engineered by swapping specific intracellular domains between the GluN2A and GluN2B subunits. In doing so, we can separate the functional contributions of individual regions, such as the intracellular tail, to ion flow and to non-ionotropic signaling. By studying receptors with these controlled domain swaps, we aim to determine which portions of the subunit structure are responsible for differences in calcium permeability, activation properties, and downstream signaling. This semester, we focused on preparing the DNA constructs necessary for expressing these receptors in Xenopus laevis oocytes for future functional testing.

The overall goal of this project is to express wild-type and chimeric NMDA receptors in Xenopus laevis oocytes and compare their ionotropic signaling properties using two-electrode voltage clamp recordings. By analyzing how domain swaps between GluN2A and GluN2B affect receptor function, we aim to better understand the molecular basis of NMDA receptor signaling. This semester, we focused on preparing high-quality plasmid DNA, optimizing restriction digests, and initiating PCR amplification of the GluN2 receptor inserts to prepare for future subcloning and expression studies.

First, upon receiving the plasmid pGEMHE-membrane-mEGFP, we transferred a sample from the backstab into a 3 mL bacterial culture, which was incubated overnight at 37 degrees Celsius for 16 to 24 hours. The plasmid includes a Xenopus laevis promoter sequence, which enables later expression in oocytes. Following incubation, we isolated and purified the plasmid DNA from the bacterial culture using a alkaline lysis mini prep protocol. To ensure the integrity and purity of the plasmid, we assessed DNA quality using agarose gel electrophoresis to check for intact plasmid structure and spectrophotometry to measure the 260/280 absorbance ratio.

Next, we performed restriction digests to prepare the plasmid for future subcloning. We used the enzyme NheI to linearize the plasmid and carried out diagnostic digests to prepare for the later excision of the GFP segment originally present in the vector.

In parallel, we grew bacterial cultures containing the DNA for GluN2A, GluN2B, ABc, and BAc constructs. Using these templates, we initiated PCR amplification with construct-specific primers to selectively amplify the inserts. PCR amplification is currently ongoing. Once complete, we will purify the amplified products and verify insert size by gel electrophoresis before moving on to the next phase of subcloning.

After the inserts are fully amplified and purified, we will digest them with restriction enzymes to create compatible ends with the plasmid vector. We will then use a DNA ligase enzyme to join the inserts and vector together, creating new plasmids that carry either the wild-type or chimeric NMDA receptor sequences. Some ligation reactions will be performed in-house, while others may be sent for commercial cloning depending on efficiency. Sequence verification will follow to confirm successful ligation.

Following sequence confirmation, we will synthesize capped RNA transcripts from the recombinant plasmids using in vitro transcription. These RNA molecules will then be injected into individual Xenopus laevis oocytes, allowing the cells to produce functional NMDA receptors for electrophysiological testing.

Two to three days after RNA injection, we will perform two-electrode voltage clamp recordings, a technique that holds the membrane potential constant while measuring ionic currents. By applying glutamate and glycine, we will evaluate receptor function based on current amplitudes, activation and deactivation kinetics, and dose-response characteristics. Comparing wild-type and chimeric receptors will help us determine how specific subunit regions influence NMDA receptor ionotropic signaling.

This semester, we focused on growing bacterial cultures, isolating and purifying plasmid DNA, troubleshooting purification and digestion protocols, and beginning PCR amplification of the NMDA receptor inserts. These steps are critical for setting up RNA synthesis, oocyte injection, and functional testing. Moving forward, we aim to complete subcloning, synthesize RNA, and characterize receptor function using TEVC recordings.

I’d like to take a moment to thank those who have been instrumental in this project.

Dr. Herin who has been an invaluable mentor in electrophysiology and molecular biology.

Dr. Dumas who has provided expert guidance on receptor signaling and chimeric constructs.

Hannah Zikria-Hagemeier who was essential in training me on plasmid preparation.

Finally, I’d like to thank the rest of the PBNJ Lab for their collective support through guidance and resources, which has been key to my growth as a researcher.

Thank you all for your help and support!

College of Humanities and Social Science College of Science OSCAR

Secretory Mitophagy Exports p53: A new pro-tumor survival mechanism.

Author(s): Layla Hasanzadah

Mentor(s): Purva Gade, Center for Applied Proteomics & Molecular Medicine

Abstract

Mitochondrial dysfunction is associated with many life-threatening illnesses, from Parkinson’s disease to malignant cancers. Cells remove damaged, aged, or stressed mitochondria through a process called mitophagy. Our team is investigating a potential pro-tumor survival mechanism cancer cells enact with the help of mitophagy initiation and subsequent export. Mitophagy initiation is sensed by the molecule PINK1, triggering the isolation and packaging of the damaged mitochondrial segment for degradation through the lysosome. Cancer cell mitophagy is triggered by elevated oxidative stress and mitochondrial DNA damage caused by hypoxia, chemotherapy, radiotherapy, and immunotherapy. In our recent study, we discovered a secretory form of mitophagy in which damaged mitochondrial segments are packaged and exported outside of the cell within PINK1+ extracellular vesicles (EV). Additionally, we found that these PINK1+ EVs export tumor suppressors (TS) phospho-P53 and Merlin along with mitochondrial segments outside the tumor cell. It has been discovered that p53 is phosphorylated by PINK1, ultimately enhancing mitophagy. We also found that Merlin, an unappreciated TS associated with neurofibromatosis, interacts with p53 through the MDM2 mediator. We hypothesize that secretory mitophagy export of TS and other mitophagy-related proteins is an adaptive mechanism of cancer cells to withstand oxidative stress and avoid apoptosis. The export of TS p53 and Merlin were analyzed in 4T1 & IOMM-Lee cells. We isolated EVs via differential ultracentrifugation followed by immunoprecipitation of purified EV samples with PINK1 and phospho-p53 to identify protein-protein interactions. Western blotting and mass spectrometry were performed to validate immunoprecipitation results and PINK1 and p53 interaction. Understanding the role of EV-mediated export of TS in secretory mitophagy can reveal novel mechanisms aiding cancer cells’ survival under oxidative stress caused by chemotherapy or other treatments, offering potential therapeutic targets for various cancers.

Audio Transcript

Mitochondrial dysfunction is associated with many life-threatening illnesses, from Parkinson’s disease to malignant cancers. Cells remove damaged, aged, or stressed mitochondria through a process called mitophagy. Mitochondria are known as the powerhouse of the cell for a reason: they help produce the majority of the energy needed to power our cells. In Parkinson’s disease, scientists have found that the process of mitophagy is impaired, leading to an accumulation of dysfunctional mitochondria within cells. On the other hand, in cancer cells, mitophagy plays a more complex role and may even be enhanced. As evidenced by my research, our lab at the Center for Applied Proteomics and Molecular Medicine found that the process may serve as a kind of survival mechanism used by cancer cells in the face of oxidative stress caused by chemotherapy or radiation.
Mitophagy initiation is sensed by the molecule PINK-1, triggering the isolation and packaging of the damaged mitochondrial segment for degradation through the lysosome. Our team discovered a secretory form of mitophagy in which mitochondrial segments are packaged and exported outside of the cell within extracellular vesicles (EVs) derived from the interstitial fluid of breast cancer tumors. Moreover, it has been discovered that the tumor suppressor molecule p53 interacts and becomes phosphorylated by PINK-1, ultimately enhancing mitophagy and carcinogenesis. Pancreatic cancer (PC) p53 mutations are associated with tumor aggressiveness. Decreased levels of intercellular p53 leads to increased genetic instability, higher tumor growth rate, and survival. Pancreatic cancer is the 3rd most fatal cancer in the U.S. due to high rates of metastasis and late diagnoses. Understanding and targeting this imbalance may result in new forms of personalized medicine for PC patients.
In terms of materials and methods, I cultured PANC-1 and BXPC-3 cell lines and incubated them for 5 days, then applied a drug called CCCP to each flask. CCCP induces oxidative stress to mimic the conditions experienced by cancer cells undergoing treatment like chemotherapy or radiation. Afterwards, I confirmed that I had protein in my sample, and used various stages of ultracentrifugation to isolate the extracellular vesicles produced by these cells. The vesicles came in 3 sizes: 2K (the largest), 10K, and 100K (the smallest). To analyze the proteins within my samples, I used Western Blotting and mass spectrometry, and used the ExoView R200 to examine and categorize the EVs used in the experiment.

Here you can see some images of me working in the lab: doing cell culture, running Western Blots, and observing my pancreatic cancer cells.

My project produced some very interesting results. I compared the relative concentrations of p53, the tumor suppressor protein, and PINK-1, the mitophagy-associated signalling molecule, and found that there is a very high and positive correlation between the export of PINK-1 p-p53 via EVs when oxidative stress is induced, indicating that p53 is degraded and exported alongside PINK-1 in EVs.Exported p53 may aid tumor progression and constitute a novel diagnostic method of non-invasively determining the mitochondrial health and p53 status within PC. PC EVs positive for phospho-p53 represent a novel diagnostic biomarker indicative of tumor stress. Targeting EV pathways in combination with oxidative stress could be a novel method of treating PC. Our lab is currently investigating if secretory mitophagy & EV export of tumor suppressors is common among other kinds of cancer, as well.

We recently published a paper on the topic of secretory mitophagy, but again, we hope to connect secretory mitophagy to the export of other tumor suppressors in future studies.

I wanted to thank my mentors and colleagues at the Center for Applied Proteomics and Molecular Medicine for their continued guidance and support, including the following people: Purva Gade, my direct mentor, Dr. Lance Liotta, Dr. Marissa Howard, Sofie Strompf, Angela Rojas, and Thomas Philipson.

I would also like to thank the GMU OSCAR URSP program and Dr. Karen Lee, as I received funding and guidance from OSCAR throughout the past semester.

Thank you very much for listening to my presentation!

College of Humanities and Social Science OSCAR

The Effects of Young Blood Plasma on NfL and GFAP following Traumatic Brain Injury Administration in Wild-type Mice

Author(s): Timothy Kochany

Mentor(s): Jane Flinn, Psychology

Abstract

Traumatic brain injuries (TBIs) are a major health concern, with there being over 200,000 TBI related hospitalizations in 2020. Repetitive mild TBIs (rmTBIs) lead to cumulative effects, worsening damage. Previous research demonstrated that young blood plasma transfusions may reverse factors of neurodegeneration in older populations, those with Alzheimer’s Disease, and those with TBIs.
Glial fibrillary acidic protein (GFAP) and neurofilament light chain (NfL) are proteins that act as biochemical markers that can be detected in blood to observe astrocyte activation and axonal integrity. Previous studies have shown that TBIs can induce neuronal death and contribute to neurodegenerative disease.
GFAP is a protein expressed by astrocytes that is involved in the injury repair of brain tissue. Overexpression of GFAP has been shown to correlate with lesion volume and TBI severity, as well as astrogliosis in releasing inflammatory mediators, such as cytokines and reactive oxygen species (ROS). This is part of the process of secondary injury during rmTBI. Neuroinflammation is a factor that contributes to neurodegeneration. This is pertinent because GFAP has been known to reflect glial responses to neuroinflammation.
Similarly, NfL is a protein found in the axon of neurons and is released into the plasma when injury or neurodegeneration is present. NfL is involved with structural support and aids in the conduction speed of action potentials. In previous studies, elevated levels of GFAP and NfL have been shown to signal the progression of neurodegeneration.

Audio Transcript

Hi everyone. My name is Tim Kochany. I’m a third-year neuroscience student at George Mason University and my project is The Effects of Young Blood Plasma on Neurofilament Light Chain and Glial Fibrillary Acidic Protein following Traumatic Brain Injury Administration in Wild-type mice. In the year 2020 alone, there have been reported over 200,000 hospitalizations related to traumatic brain injuries. This is a major world health concern because traumatic brain injuries have been known to lead to neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease, as well as cognitive decline. Previous research has demonstrated that plasma from young mice injected into older mice has seemed to rescue deficits induced by traumatic brain injuries. My research focuses on two proteins that are involved in this process: Glial Fibrillary Acidic Protein, or GFAP, and Neurofilament Light Chain, or NfL. Glial Fibrillary Acidic Protein is a protein that is expressed by astrocytes and is involved in the injury repair process of the brain. Similarly, Neurofilament Light Chain is a protein found in axons, which is a part of the neuron that helps propagate the signal from one neuron to the next, and overexpression of this protein has been shown to signal the progression of neurodegeneration. Repetitive mild traumatic brain injuries lead to cumulative effects, worsening brain damage. Prior to the current study, plasma injections and saline injections have been administered and the mice have been euthanized and plasma samples have been collected. There are four groups of mice: TBI plasma, TBI saline, sham plasma, and sham saline. Sham is if no TBI has been administered. For the procedure, I am going to be looking at and I’m going to be using Enzyme-linked Immunosorbent Assay or ELISA to detect protein quantities in the plasma from these mice. For this process, antibodies for the specific protein are going to bind to that protein and then we are going to detect protein concentrations by shining a specific wavelength of light through the sample. My hypothesis is that traumatic brain injuries will increase levels of both GFAP and NfL, with the groups receiving plasma injections slightly lowering these concentrations. This research has significant implications for the study of neurodegeneration and public health relating to traumatic brain injury treatment. Unfortunately, no results are available at this time, as I’ve had errors with our ELISA microplate reader malfunctioning to collect data. However, data will be available at the OSCAR poster celebration on May 6. If you would like to see a full list of my works cited, you can scan this QR code here.

College of Humanities and Social Science OSCAR

Is Executive Function Similarly Related to Behavioral Outcomes in Typically Developing and Neurodiverse Children? A Meta-Analysis.

Author(s): Armita Dadvar

Mentor(s): Sabine Doebel, Psychology

Abstract

Executive functions (EFs) are a set of neurocognitive processes that support goal-directed thought and action. EFs are essential to child development, especially in its concurrent and longitudinal relation to both academic achievement (Allan et al., 2014; Blair et al., 2015; Spiegel et al., 2021) and school readiness (Blair & Razza, 2007). Research suggests that children diagnosed with neurodevelopmental conditions show greater impairment in EF compared to their neurotypical peers (All, et al., 2024). The project investigated the relation between EF in young children and behavioral outcomes by conducting a mixed-effects meta-analysis. It explored whether the relation between child EF and behavioral outcomes varies depending on whether children are typically developing or have clinical, learning, or behavioral diagnoses. Detailed inclusion criteria were established and we conducted a systematic literature search across four academic databases. Abstract screening was performed to identify papers that meet the inclusion and exclusion criteria for the study. All eligible studies were identified, and the data was coded and analyzed with the help of MetaReviewer. For statistical analysis, we used CHE modeling through the metafor and clubSandwich packages on R-Studio. Results indicated no significant association between executive function and internalizing or externalizing symptoms, but a small significant trend linking better EF to fewer hyperactivity and inattention behaviors. No significant differences were found between neurodiverse and neurotypical children. However, better EF was significantly related to fewer externalizing problems in children with ADHD, and younger age was associated with a stronger EF–behavior relationship. These findings suggest that EF may be a modest intervention target for early behavioral difficulties, with future research needed to explore diagnostic-specific patterns and increase study representation for neurodivergent populations.

Audio Transcript

Hello everyone, my name is Armita Dadvar and this presentation is on my Honors Psychology Thesis titled “Is Executive Function Similarly Related to Behavioral Outcomes in Typically Developing and Neurodiverse Children?” which I completed with my advisor Dr. Sabine Doebel. Have you ever heard of the term executive function? Executive functions are the set of neurocognitive processes that help us engage control over our thoughts and actions, manage and retain the information in our brains, preventing undesirable thoughts and behaviors while prioritizing ideas and actions that are aligned with personal objectives. You might be thinking, why is executive function important to study in childhood? It is important because research has shown that executive function rapidly develops through childhood and essential to child development. It even has concurrent and longitudinal relation to academic achievement and school readiness. It also may be a key path through which socioeconomic status influences academic outcomes. Lastly, It is even believed to be more malleable than IQ and other general cognitive capacities and thus a good target for interventions. Over many years, numerous tests have been conducted by researchers to carefully evaluate EFs as they arise through childhood. Although we understand that EF is is related to many behavioral outcomes in typical developing children, we do not know if these relations are similar for children who are neurodiverse or have learning, behavioral, or clinical diagnoses that implicate brain areas supporting EF. The current project investigated, via a meta-analysis, the relation between EF in young children and behavioral outcomes, and whether the relation between child EF and outcomes varies depending on if children are typically developing or neurodiverse. Our research question is: Does the relation between child EF and behavioral outcomes vary depending on the population under consideration? We hypothesized that there will be stronger, more consistent executive function/outcome relations in neurodiverse vs neurotypical samples. For this meta-analysis, we were specifically interested in early childhood, executive function and related moderators, key moderators of population, socioeconomic status, age and gender and lastly three main behavior outcomes or externalizing, internalizing, and inattention/hyperactivity. in terms of methods, we first finalized the inclusion and exclusion criteria, then we conducted searches across 4 different bases. After collecting results from those searches, we screened a smaller percentage of abstracts and determined which do or do not meet the inclusion criteria. All eligible abstracts moved on to full text coding through Metareviewer. For statistical analysis, we used CHE modeling to account for dependency among effect sizes. We ran the models with the rma.mv() function from the metafor package in R and calculated robust test statistics using the coef_test() function from the clubSandwich package. This approach gives more accurate variance estimates and properly handles the fact that some studies contributed multiple effect sizes. We found no significant relationship between executive function and internalizing and externalizing symptoms. However, there was a small-to-moderate significant trend suggesting better EF might be linked to fewer hyperactivity and inattention symptoms.In terms of our hypothesis, we did not find meaningful group differences between neurodiverse and neurotypical children in these relationships.When exploring moderators, we found better EF was significantly related to fewer externalizing symptoms in children with ADHD. However, no strong associations were found for hyperactivity/inattention in ADHD or for group differences between ADHD and typical samples. We also found that the EF-behavior relationship became weaker as children got older. No effects were found for the sex moderator and SES could not be analyzed due to too much missing data. Our results suggest that better executive function may be modestly linked to fewer hyperactivity and inattention symptoms in young children, consistent with previous research. Future research should expand the number of studies, especially for neurodivergent children, examine specific diagnoses separately, and evaluate potential publication bias to strengthen conclusions. Thank you so much to my advisor, Dr. Sabine Doebel, the Developing Minds Lab, and my committee members Dr. Jennifer Brielmaier and Dr. James Maddux for their expertise and guidance throughout this project. Thank you to OSCAR for the funding of this project through the URSP. Lastly, here are my references and thank you all for listening.

College of Humanities and Social Science OSCAR

Changes in BDNF in a mouse model of Alzheimer’s Disease with APOE4 and Tau compared to wild type mice

Author(s): Jasmine Mendoza

Mentor(s): Jane Flinn, Psychology

Abstract

Alzheimer’s Disease (AD) is a neurodegenerative disease characterized by two biological components: an increase in both amyloid beta (Abeta) and hyperphosphorylated tau (p-tau). According to the dual pathway hypothesis, Abeta and p-tau accumulate independently yet simultaneously. A possible mechanism for the dual pathway hypothesis is Apolipoprotein E (APOE), a protein which plays a central role in neurodegenerative diseases such as AD. The E4 isoform of APOE is most commonly associated with a higher risk for late-onset AD, though recent research indicates APOE4 may play a neuroprotective role when not in the presence of Abeta. Another protein that plays a neuroprotective role is brain-derived neurotrophic factor (BDNF), which is essential for neural plasticity and therefore learning and memory. Decreased amounts of BDNF are correlated with more severe cognitive deficits associated with AD, and APOE4 and Tau have previously been found to reduce BDNF levels. However, there has yet to be a study that examines the effects of APOE4 and Tau on BDNF in the absence of Abeta. The present study aims to fill this research gap using Western Blots on 4 different genotypes of mice (E4 x Tau, Tau, E4, and wild type) to target BDNF.

Audio Transcript

Hello everyone, my name is Jasmine Mendoza and my mentor is Dr. Jane Flinn, and my project is titled “Changes in BDNF in a mouse model of Alzheimer’s Disease with APOE4 and Tau compared to wild type mice.”

To begin, I’d like to define some key terms in my project. Alzheimer’s Disease is a neurodegenerative disease that affects learning and memory. Late-onset Alzheimer’s Disease is typically diagnosed in individuals age 65 and up, while early-onset Alzheimer’s Disease, which is much less common, is typically diagnosed in individuals below the age of 65.

Brain-derived neurotrophic factor, or BDNF, is an essential protein for learning and memory, as it promotes neuronal growth and plasticity. In contrast, Apolipoprotein E, or APOE for short, is a protein that helps metabolize fats, and there are three different alleles of the APOE protein. There’s APOE2, APOE3, and APOE4, with APOE4 being commonly associated with late-onset Alzheimer’s Disease, and it’s considered a high risk-factor for the disease.

Finally, the two key biological components of Alzheimer’s Disease are the peptide amyloid beta, or Abeta, which accumulates into plaques, and a version of the protein tau which has an excessive amount of phosphate groups attached to it, which is referred to as hyperphosphorylated tau, or p- tau, and this hyperphosphorylation causes it to accumulate into tangles.

The previous literature on Alzheimer’s Disease has largely found that APOE4 has detrimental effects on AD patients, usually exacerbating the cognitive deficits associated with the disease. For instance, the dual pathway hypothesis proposed by Small & Duff suggests that the accumulation of Abeta and p-tau happens independently but still parallel to each other, which eventually leads to neuronal death and cognitive decline, and that this accumulation may be facilitated by APOE4. Likewise, APOE4 has been found to reduce BDNF levels, which results in more severe cognitive deficits, as BDNF is a neuroprotective protein, therefore the reduced amounts of it are associated with more severe cognitive deficits, as found by Laske et al. in 2011. However, a recent study done by a graduate student who is also in my mentor’s lab, found results that suggest APOE4 and Tau may play a neuroprotective role when they are not in the presence of Abeta. The current literature has not yet examined how APOE4 and Tau interact with BDNF in the absence of Abeta, so this study aims to address that gap in the literature, and is also a continuation of that 2024 study by Booth in my mentor’s lab.

So, because this study is a continuation of the previous Booth study, the brains used come from mice that were separated into four different genotype groups and two different metal ion supplement groups, as those were the groups used in the Booth 2024 study. The four different genotype groups are those with APOE4, those with Tau, those with both APOE4 and tau, and wild type mice without either APOE4 or tau. The two metal ion supplement groups are those with zinc and those without any metal ion supplement. So, this study will use Western Blots, which is a technique which targets specific proteins and isolates them from other proteins in biological samples. And, the proteins are targeted through the use of antibodies which bind to the target protein and are prevented from binding to any other proteins by a process called gel electrophoresis. The primary antibodies that we’ll be using in this study are BDNF, of course, and Glyceraldehyde 3-phosphate dehydrogenase, or GAPDH, which will be used as a loading control. The secondary antibody will be mouse anti-rabbit, which will bind to the primary antibody and make it easier to visualize and quantify BDNF. And then the data will be analyzed in SPSS. As for the possible implications of this study, the findings of Booth (2024) were unlike those of previous studies examining the effects of APOE4 in Alzheimer’s Disease, as most previous studies, as we’ve discussed, have found negative effects of APOE4, while the results of his study actually found a possible neuroprotective effect that APOE plays. In his study, Booth does suggest that the ages of the mice used may have been a mediating factor in this effect, because the mice were younger, so it’s possible that APOE4 only plays a neuroprotective role in younger individuals. Thus, a future study could possibly examine the effects of APOE4 on BDNF in different age groups. Nonetheless, what we are hoping to find with this study is more information on the interaction of APOE4, Tau, and BDNF in Alzheimer’s Disease mice, which could eventually lead to new possible treatments involving APOE4 and Tau.

Finally, I would like to thank my mentor, Dr. Jane Flinn, as well as everyone in the Flinn lab, and I would also like to thank Dr. Karen Lee and URSP as a whole for providing funding and guidance throughout this project.

And here are my references. Thank you for your time, and I hope you enjoyed hearing about my study.

College of Humanities and Social Science College of Science OSCAR

Use of a Novel In-Vivo Acetylcholine Sensor, GRABACh3.0, to Quantify the Temporal Dynamics of Acetylcholine (ACh) Release in the Cornu Ammonis 1 (CA1) Hippocampus Sub-Region

Author(s): Muhammad Shah

Mentor(s): Holger Dannenberg, Interdisciplinary Program in Neuroscience

Warning Video Contain Graphic Images

Abstract

Ongoing research aims to uncover how the brain processes spatial information, with the
long-term goal of informing therapies for spatial memory dysfunctions seen in
dementias. One key neuronal substrate is the place cell—neurons located in the Cornu
Ammonis 1 (CA1) subregion of the hippocampus that exhibit spatially tuned firing.
Recent studies suggest that acetylcholine (ACh), released into CA1 from the medial
septum, modulates place cell activity. Investigating how ACh modulates hippocampal
circuits during real-time behavior is essential to understanding its role in spatial
encoding.

The recent development of the fluorescent ACh sensor GRABACh3.0 enables real-time
measurement of cortical ACh activity in animal models. Using this sensor, we aim to
quantify the temporal dynamics of ACh release in the CA1 region during free-roaming
behavior in mice. To accomplish this, we will perform a stereotaxic injection of an
adeno-associated virus (rAAV) encoding GRABACh3.0 into CA1, followed by
implantation of an optic fiber above the injection site to permit fluorescence-based
recording via fiber photometry.

After allowing two weeks for sensor expression, a fiber photometry system will be used
to deliver constant excitation light specific to the sensor and record resulting
fluorescence during 15-minute free-roaming trials in a novel boxed environment.
Simultaneously, mouse velocity will be estimated using DeepLabCut, a markerless AI-
based pose estimation tool. Fluorescence signals will be synchronized with velocity data
to assess their temporal relationship.

Preliminary data revealed a positive correlation (r = 0.60) between ACh sensor
fluorescence and mouse velocity during free-roaming trials—a relationship consistent
with prior literature, supporting the validity of our recorded ACh signal. Next, we aim to
replicate this model and examine ACh release in CA1 during behavioral assays of
novelty and familiarity, to further investigate the neuromodulatory role of ACh in spatial
encoding.

Audio Transcript

Hello

My name is Muhammad, and my project is “Using a Novel In-Vivo Acetylcholine Sensor, GRABACh3.0, to Quantify the Temporal Dynamics of Acetylcholine (ACh) release in the Cornu Ammonis 1 (CA1) Hippocampus Sub-Region.”

In 1971, John O’Keeffe, a Nobel Prize winning neuroscientist, probed the electrical activity of hippocampal CA1 pyramidal neurons with electrodes in mouse models. He noticed that as the mouse traversed the environment, certain populations of neurons would increase firing rates in select regions of the box, which is now known as spatially tuned firing.

Discovery of these cells offered a new insight into how individual neurons encode space, and these CA1 neuron types would cleverly be named place cells! With this discovery came renewed interest in understanding the place cells within the CA1 region, with the goal of further uncovering the mechanisms of spatial cognition to inform future treatments for spatial amnesia in patients with, for example, Alzheimer’s disease.

Recently, it was discovered through retrograde tracing that Acetylcholine or ACh is released into CA1 region from distant cholinergic afferents from the medial septum. It is believed that these cholinergic afferents play a role in either directly stimulating place cells or stimulating other surrounding interneurons to inhibit place cells in the CA1 region.

Based on the multimodal modulation of place cells by ACh, understanding the release dynamics of ACh in real-time within the CA1 during behavior is of great interest.

To accomplish this, we used a novel ACh sensor called GRABACh3.0. Its major feature of interest is its binary nature, where it is capable of emission when ACh is bound but is incapable of emission when ACh is not bound. Thus, by using this sensor, the real-time neurodynamics of ACh within neuronal circuits can be observed.

To express the ACh sensor in mouse models, it is first packaged into a viral vector and then injected into the CA1 region, as seen here. We were able to confirm the expression of the sensor through immunohistology, shown here.

A fiber optic receiver is then implanted into the same location within the CA1 as the virus injection to measure the expressed ACh sensor. Then, a fiber photometry system is used to stimulate the sensor. Fiber photometry is a powerful technique that allows both excitation light, specific to the sensor, and control light to be transmitted through the same wire into the mouse fiber optic. It also allows, from the same input wire, to receive and record any resulting emission from either excitation or control light source, respectively, thus separating their influence on fluorescence within the sample. The fluorescence data is then converted into an electric signal using a phototransducer within the system.

Here is the system on the left where you can see both of the excitation cords that go into the sample and the emission cord coming back from the sample. We record fiber data for 15 minutes in a boxed environment. We also sync the fiber system with a bird’s eye video recording of the mouse to see how ACh levels correlate with the velocity of the mouse during the free roaming trials.

This correlation is found between ACh levels and velocity data to confirm whether we truly see an ACh signal from the mouse, as prior literature has established this positive correlation. After subtracting the control signal from the ACh signal and finding the velocity data using an AI markerless pose estimator, DeepLabCut, we obtained these preliminary results.

The graphs on the left show the isolated ACh signal, the velocity data, and their superimposition. Zooming in, we can see at specific time points when the animal increases its running speed, as marked by the arrows, that ACh activity tends to increase along with it. Furthermore, the overall correlation between the two variables was 0.60, which is very unlikely to be due to chance, especially over almost 45000 data points.

These findings were very exciting, but we face the next challenge of getting replicable results. The current findings come from our most recent and successful mouse, which resulted from a series of trial and error from previous mouse models using the sensor, as it is our lab’s first time using this specific sensor within the CA1. We are currently waiting on a new cohort of mice to see if we can consistently get results similar to those seen recently.

If we achieve consistency, we hope to use our standardized procedure and combine it with other techniques, such as optogenetics or spatial behavioral assays, in the future.

I want to give a big thank you to URSP for funding, Dr. Holger Dannenberg for his mentorship, and the Spatial Cognition Lab team for their support—this project would not have been possible without them.

College of Humanities and Social Science OSCAR

The Effect of Ionotropic and Non-ionotropic NMDAR Signaling Domains on Postnatal Development of LTD Using Chimeric GluN2 Mice

Author(s): Bryce Sullivan

Mentor(s): Theodore Dumas, Neuroscience

Abstract

The hippocampus, which is a brain region essential for information processing, learning, and memory formation, undergoes critical periods of heightened experience-dependent changes to neuronal structure and function (plasticity). N-methyl-D-aspartate receptors (NMDARs) are necessary for this process in excitatory hippocampal synapses through synaptic strenghening (long-term potentiation, LTP) and weakening (long-term depression, LTD). NMDARs have been shown to behave both ionotropically and nonionotropically, and that specific domains on the GluN2A and GluN2B subunits contribute to synaptic plasticity. In mice, the third postnatal week (~three years old in humans) marks the onset of mature learning and memory and is the result of a shift of dominance from GluN2B to GluN2A subunits. In this project, the impact of individual NMDAR GluN2 subunits signaling streams on the hippocampal development of LTD. This was done using chimeric that contain either GluN2A or GluN2B with the carboxy terminal domains (CTDs) swapped (i.e. Glu2A-BCTD and GluN2B-ACTD), which allows isolation of the ionotropic and nonionotropic signaling properties of each GluN2 subunit. A 1 Hz stimulation was used for 15 minutes to induce LTD in CA1 of hippocampal slices of these chimeric constructs and wild-type (WT) and the field excitatory post-synaptic potentials (fEPSPs) were recorded. While more data needs to be collected and analyzed, these experiments will provide a better understanding of how ionotropic and nonionotropic NMDAR signaling domains regulate the late postnatal development of LTD.

Audio Transcript

Hello everyone! My name is Bryce Sullivan, and my projected is looking at the effect of ionotropic and non-ionotropic NMDAR Signaling Domains on Postnatal Development of LTD Using Chimeric GluN2 Mice

The hippocampus, named after its seahorse-like shape in humans, is an essential brain structure for spatial learning and memory in adults. Development of the hippocampus occurs in the late postnatal period and coincides with the emergence of adult-like spatial learning and memory abilities at around three years of age in humans, or three weeks of age in rodents.

Synapses are the electrochemical junctions between neurons that allow for neural circuits to be built and to process information. The ability of excitatory synapses in the hippocampus to alter their function with new experiences and create neural substrates of memories is referred to as synaptic plasticity.” The final maturation process of the hippocampus involves modifications in synaptic plasticity.

In our lab, we study the NMDA receptor, NMDARs, which are essential for activity-dependent plasticity at excitatory synapses. The prominent forms of synaptic plasticity induced by NMDAR activation are long-term potentiation, LTP, and long-term depression, LTD. So, when a synapse is activated at high frequencies, NMDARs respond by sending signals to strengthen that synapse, which is LTP. However, when a synapse is activated at lower frequencies, NMDARs send a signal to weaken that synapse, which is LTD.

NMDARs are ion channels that allow calcium to enter the activated cell. It is well known that calcium activates synaptic plasticity processes. However, in 2013, it was discovered that NMDARs produce signals that are not related to calcium, that are mediated by the intracellular portion of the NMDAR subunits.

To study separate influences of calcium-dependent and calcium-independent signals, our lab created chimeric NMDAR subunits. I am using mice that express chimeric NMDAR subunits to investigate which NMDAR signaling pathways underlie the development of LTD. My electrophysiological recordings take place on postnatal day (or P) 17 to 19 and P22 to 24. These two age groups represent the immature hippocampus (younger than three postnatal weeks) and the mature hippocampus (older than three postnatal weeks).

My project’s primary three methodologies consist of genotyping, hippocampal slice prep, and hippocampal slice recordings of field excitatory post-synaptic potentials (fEPSPs) in CA1. Genotyping consists of taking small tail snips of our mice, DNA precipitation to isolate a DNA sample from that tail, running a polymerase chain reaction (or PCR) to isolate and amplify our chimera genes (if present), and then running a gel electrophoresis to find their genotype.

Hippocampal slice preparation is as it sounds; sacrificing the mouse, dissecting and isolating the hippocampus, and cutting the hippocampus into slices that are about a half a millimeter thick. These slices can be maintained alive in a recording chamber for many hours.

To record from hippocampal slices, we use two electrodes as this sample image (A) shows here. The bipolar electrode here is a platinum-iridium wire that activates the input neurons that project to area CA1. Synaptic responses are recorded in area CA1. The waveform in B represents an excitatory synaptic potential. The dotted line shows how we measure the slope of this response.

When LTP is induced, the EPSP gets larger. When LTD is induced, the EPSP gets smaller. For our LTD protocol, we use a 1Hz stimulation for 15 minutes.

It is well known that low frequency stimulation elicits LTD to a greater extent in immature mice. However it is not known if this reduction in LTD with increasing age is due to changes in calcium-dependent or calcium-independent NMDAR signaling. Performing LTD experiments in mice that express chimeric NMDAR subunits allows for testing of these two different possibilities.

I would finally like to thank OSCAR for providing me funding for my project with the URSP program and Dr. Dumas along with the rest of the PBNJ lab for their support and mentorship. This is not the present PBNJ lab, but I felt as though this photo from before 2015 was appropriate

And here are my references

College of Humanities and Social Science OSCAR

Binaural Beats on Attention

Author(s): Alisha Jane Hiskey

Mentor(s): Martin Wiener, Neuroscience

Abstract

Binaural beats is a non-invasive and inexpensive nonpharmaceutical potential treatment for disorders of attention, among many others. Binaural beats are a phenomenon that occurs when two sounds with slightly different tones are played to each ear. The brain synthesizes these varied amplitudes into one cohesive sound that oscillates back and forth. There has been some recent research into the effectiveness of binaural beats in improving areas like sleep, meditation, memory, and attention.
The Brainwave Entrainment Hypothesis suggests that after being exposed to a specific frequency of sound, the brain will adjust and mirror that activity on a similar frequency (Ingendoh, 2023.) A 22 study meta-analysis adds to the growing evidence that binaural-beat exposure is an effective way to affect cognition”¦ without prior training” (Garcia-Argibay, 2019.) However, more work can be done as there is a lack of studies examining the effects of binaural beats on autonomic activity in relation to cognition” (Chee, 2024.)

Audio Transcript

Hello, my name is Alisha Hiskey. I am pleased to share my presentation on binaural beats. Binaural beats are a phenomenon that occurs when two sounds with slightly different tones are played to each ear. The brain synthesizes these varied amplitudes into one cohesive sound that oscillates back and forth. There has been some recent research into the effectiveness of binaural beats in improving areas like sleep, meditation, memory, and attention. This project works to investigate the surrounding effects of various binaural beats recordings on attention. The Brainwave Entrainment Hypothesis suggests that after being exposed to a specific frequency of sound, the brain will adjust and mirror that activity on a similar frequency (Ingendoh, 2023.) A 22 study meta-analysis adds to the growing evidence that binaural-beat exposure is an effective way to affect cognition over and above reducing anxiety levels and the perception of pain without prior training” (Garcia-Argibay, 2019.) However, more work can be done as there is a lack of studies examining the effects of binaural beats on autonomic activity in relation to cognition” (Chee, 2024.) In response to this call to action, a task called a temporal bisection has been created that evaluates aspects of attention such as reaction time, precision, and response bias. Three groups are going to be divided and either a binaural beats frequency or pink noise (control) are played for the participant 10 minutes before and during then the task. During one cycle, the task requires the participant to respond short or long” to a stimulus shown 300-900ms. Each frequency that is played can be conducive to various brain states like one of sustained attention and cognitive power when entrained in the beta or gamma range. The shifting from high energy/anxious states into more relaxed and focused ones could help people with ADHD, ADD and other related attention deficit disorders. After this experiment, there can be added information on the effectiveness of binaural beats in the world of music therapy and beyond. I would like to thank OSCAR and the Undergraduate Research Scholars Program for their support and funding. I would also like to thank Dr. Karen Lee and Dr. Martin Wiener for their guidance during this project. Thank you for your time!

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