The Impact of Prenatal Marijuana Exposure on the Developing Human Brain
Advisor: Dr. Zeynep Araci
Internship Location: University of California San Francisco Parnassus Campus (505 Parnassus Ave, San Francisco, CA 94143)
Onsite Mentor: Tanzila Mukhtar
As cannabis (more commonly known as marijuana) legalization spreads, especially in the United States, more people are using it without fully understanding the risks, with up to 19% of pregnant individuals in California using cannabis, believing it is safe. While many view marijuana as a safer alternative to prescription meds or alcohol, it can actually have serious consequences for brain development, especially in the early stages of life. For my project, I’m focusing on how prenatal exposure to cannabis, specifically tetrahydrocannabinol (THC) and Cannabidiol (CBD), affects brain development. To explore this, I’ll be using cortical organoids, which are 3D models of the brain created from stem cells. These organoids mimic human brain development and allow me to study how exposure to THC and CBD impacts brain cells, particularly those responsible for brain signaling. With guidance from my advisor, Dr. Tanzila Mukhtar, I’ll be examining how these compounds affect the growth and function of neurons, especially excitatory and inhibitory neurons that are critical to brain activity. By the end of this project, I hope to uncover the specific ways cannabis affects fetal brain development. This research will help provide clearer guidance for public health recommendations, potentially shaping policies that inform expecting parents and healthcare providers about the risks of cannabis use during pregnancy. Ultimately, it will contribute to the ongoing conversation about the safety of cannabis during pregnancy, leading to better informed decisions for both individuals and healthcare systems.
Project Blog
Post 1: Blog Zero Post
Hi everyone! Welcome to my first blog post! I’m really excited to start this journey with you as I take on my Senior Project, where I’ll be exploring the effects of prenatal cannabis exposure on brain development. Stick with me as I dive into a topic that’s not only personal but also incredibly important for public health.
Growing up in California, I’ve seen cannabis go from being illegal to becoming more widely accepted and even legalized. With this shift, I noticed something that really caught my attention: how little people understand about the impact cannabis might have on developing babies. It’s something I’ve thought a lot about, especially when I see so many people turning to it without fully realizing the risks. Personally, I’ve always been someone who questions the status quo and seeks the truth behind things, so when I learned about the potential neurodevelopmental risks of cannabis, I knew I wanted to study it further.
What drew me to this project was the opportunity to explore a question that directly affects people in my community, pregnant individuals who are exposed to cannabis. I want to understand how THC and CBD, two of the most common compounds in cannabis, can alter brain development in the early stages of life. As I learned more about this issue, it became clear to me that there’s a gap in knowledge that needs to be filled, and I want to help change that.
I’ll be working under Dr. Tanzila Mukhtar at UCSF’s Parnassus Lab, using advanced techniques like single-cell sequencing and calcium imaging to look at how these compounds impact brain cells. My goal isn’t just to conduct research for the sake of science; I want to bring real, tangible information that can make a difference. I know that if we can better understand the effects of prenatal cannabis exposure, we can give expecting parents and healthcare providers the tools they need to make better-informed decisions.
This project means a lot to me because it combines both my curiosity and my desire to help others. It’s not just about adding to the science but also about providing people with the information they need to protect their children and families. As cannabis becomes more common, it’s crucial to make sure we understand all the risks involved, especially the ones we might not even see coming.
Stay tuned for my next post, where I’ll dive deeper into the research process and share what I’m learning along the way. Thanks for reading, and I can’t wait to share more with you all!
Week 1: Learning the Basics
It's great to have you all here again!
This week has been an absolute adventure as I dove headfirst into some of the coolest techniques that will help me unravel how prenatal marijuana exposure affects prenatal development. Imagine stepping into a world where every cell tells a story, that’s been my reality in the lab these past few days.
I kicked things off by getting hands-on with immunostaining. If you haven’t heard of it, think of it as a way to tag specific proteins within tissue samples so you can actually see what’s happening at a molecular level. It felt a bit like magic, watching those markers light up and reveal the hidden details in the cells. This technique is a game-changer for spotting the subtle changes that might occur due to prenatal marijuana exposure. I won't lie, the learning curve was steep. It took me a while to wrap my head around all the different antibodies and their specific functions. I found myself buried in papers and my notes trying to understand which antibody was best for targeting a particular protein in different regions of the developing brain. To keep everything straight, I even had to create a table. This table listed each antibody, its target protein location, and notes on the functions of the factors they were binding to. Although it was challenging, putting everything into a table helped me organize my thoughts and provided a quick reference during those long lab sessions. There was a lot, and you don't need to know all that, so here's just a part of it.
Antibodies | Location | Related Function
Z01 | Tight junctions of epithelial/endothelial cells | Maintains cell–cell adhesion and barrier integrity
SCGN | CGE Dervied Interneurons-Cytoplasm | Calcium-binding protein involved in modulating neuronal signaling
Nestin | Neural Stem/Progenitor Cells-Radial Proteases | Intermediate filament marker for neural progenitors
YAP1 (TF) | Blodd Vessels and Radial Glia-Nucleus and Cytoplasm | Transcriptional co-activator; key effector in the Hippo pathway
Olig2 (TF) | Nucleus of Oligodendrocytes | Regulates oligodendrocyte differentiation and motor neuron development
TBR1 (TF) | Nucleus of Postmitotic Coritcal Neurons-Deep Layer, Layer 6, Layer 1 | Transcription factor critical for cortical neuron differentiation
CTIP2 (TF) | Nucleus in Deep Layer Cortical Neurons- Deep layer, Layer 5 | Transcription factor (BCL11B) involved in neuronal subtype specification
SATB2 (TF) | Neurons in Uper Layer Cortical Regions-Upper layer, layer 4 | Regulates gene expression during cortical development and connectivity
SOX5 (TF) | Nucleus-Deep Layer, Layer 5 | Transcription factor involved in neuronal differentiation
NR2F2 (TF) | Nucleus-CGE Dervied Interneurons | Nuclear receptor implicated in cortical and vascular development
EGFR | Cell Membrane-Oligodendrocytes, Outer Radial Glia, | Receptor tyrosine kinase that mediates cell growth, survival, and proliferation
DCX | Neuronal Cytoplasm-Neurons, IPSCs | Microtubule-associated protein; marker for migrating neuroblasts
GFAP | Astrocyte Cytoskeleton | Intermediate filament; marker for astrocytes and glial activation
DAPI | Nucelus-Binds DNA | Fluorescent stain for DNA; used for nuclear visualization
MAP2 | Neuronal dendrites | Stabilizes microtubules; dendritic marker in mature neurons
SOX2 | Nucleus of neural stem/progenitor cells | Transcription factor that maintains stem cell properties-maintains the pluripotency of early embryonic cells
Synaptophysin | Presynaptic terminals | Synaptic vesicle protein; marker for synapse formation
PAX6 | Nucleus of neural progenitors | Transcription factor critical for neurogenesis and eye development
Ki-67 | Nucleus of proliferating cells | Marker of cell proliferation
HuC/D | Neuronal cytoplasm and nucleus | RNA-binding proteins; marker for postmitotic neurons
Activated Caspase-3 (pCASP3) | Cytoplasm of apoptotic cells | Effector caspase; indicator of apoptosis
Next, I embarked on a mini tour through the developing brain. What I mean by that is it was time for me to learn all the basics. I started at the ventricular zone, where the action is nonstop with neural stem cells dividing left and right. Then, I moved on to the marginal zone and the cortical plate, areas that are crucial for forming the cerebral cortex. Seeing these regions in action was eye-opening and really helped me appreciate the intricate dance of brain development, and how easily it can be influenced by external factors.
But wait, there’s more! I had the chance to sit in on a fascinating journal discussion about organoids. These lab-grown, tiny versions of brain structures offer an incredible window into early neural development. Specifically the discussion was about the different limits and factors that are controlling organoid development. Although I didn’t understand most of it at first, I did catch on to some things, but it’s probably going to take a lot more for me to understand all of it, most likely through reading a bunch of papers.
On top of all that, I also got my hands on some high-tech tools that are quickly becoming my lab best friends. I learned how to use the Zen software for microscopy, capturing those high-resolution images of my immunostained samples felt like taking a snapshot of the future. Then, I dove into Imaris software for image analysis, which lets me zoom in and quantify the intricate details of the cellular landscape with incredible precision. Honestly, playing around with these tools has me feeling like I’ve unlocked a whole new level of lab superpowers.
All in all, this week has been all about laying down a solid foundation for my research. Every new technique and tool is another piece of the puzzle, bringing me closer to understanding the subtle yet significant ways prenatal marijuana exposure can alter fetal brain development. I’m both challenged and inspired by the complexity of this work, and I can’t wait to see where this journey takes me next.
Thanks for joining me today. I’m already buzzing with excitement for next week’s events. Stay curious and talk soon!
Sources:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4701311/
https://pubmed.ncbi.nlm.nih.gov/31667550/
https://www.nichd.nih.gov/health/topics/marijuana
https://www.zeiss.com/microscopy/us/products/microscope-software/zen.html
https://imaris.oxinst.com/
Week 2: The Dark Room
Hey everyone, welcome back to another chapter of my research journey! This week, I’ve truly been living in the lab, early mornings, terrible lighting, and a whole lot of science in the dark room. Let’s dive into the nitty-gritty details, shall we?
Imagine stepping into a room where the only light comes from the soft glow of your microscope screen and your computer. That’s been my reality this week as I worked with the Zen software for microscopy. I was examining slides from gestation weeks 18 to 22, each meticulously prepared with one of four treatments: THC, CBD, Fentanyl, or a Control (using methanol). It’s a bit surreal, knowing that every slide is a tiny window into how these substances might influence prenatal brain development.
Using Zen, I was able to adjust every parameter, from focus and brightness to contrast, to ensure I captured every minute detail of the tissue architecture.But here’s where it gets really fascinating: I wasn’t just taking pretty pictures. I was on a mission to track the expression of some key genes that serve as molecular markers of brain development. I took 20X photos with the software carefully adjusting the signal of each gene we were tracking to get the best possible images I could. I zeroed in on several critical proteins:
SOX2: This one’s essential for maintaining neural stem cell identity. Seeing its expression tells me where the brain is still in its “undecided” state.
EOMES: A marker that signals the early steps of neuronal differentiation. Its presence helps me gauge how stem cells are transitioning into neurons.
NEUN: This protein is like a stamp of approval for mature neurons, an essential indicator for neuronal maturation.
HOPX: Involved in the regulation of stem cell differentiation, it gives insight into early developmental shifts.
Ki67: A classic proliferation marker, perfect for identifying which cells are actively dividing.
GFAP: A glial marker, pointing to the development of astrocytes.
CTIP2 and SATB2: These markers are crucial for distinguishing between different neuronal subtypes in the cortical plate.
I specifically focused on these markers in two regions of the brain: the Outer Subventricular Zone (OSVZ) and the cortical plate (CP). That is because those are the two regions safest for microscopy as it was least likely that the sections would be broken there, so I would be able to get a more complete and clear image. For example, the edges of each section would be the ventricular zone on one side, and the marginal zone on the other. During sectioning or slicing each sample might be accidentally made in a way that part of either the VZ or MZ would be missing. In order to get the greatest results it was safer to take images near the middle of the samples, those places being the OSVZ and the CP. It was exhilarating to see the unique patterns in each region, like deciphering a complex map where each marker is a landmark guiding me to understand cellular changes induced by THC and CBD.
While my eyes were glued to the screen in the dark room, my brain was busy piecing together the broader picture. I spent a lot of time reviewing the basics of genes related to THC and CBD signaling. For instance, I dug into how these cannabinoids might influence gene expression patterns that are critical for neural development. It turns out that even subtle shifts in the expression of genes like Sox2 or Eomes can have ripple effects on brain maturation and organization. This intersection of genetics and pharmacology is as challenging as it is fascinating, and every new piece of information felt like discovering a secret passage in the maze of prenatal development.
I took hundreds of high-resolution photos of the slides, each one capturing a different angle or a unique pattern of protein expression. These images aren’t just snapshots; they’re data waiting to be analyzed with Imaris software. I can’t wait to dive into the 3D reconstructions and quantitative analyses that IMARIS promises, as it will help me build a clearer picture of how these treatments affect cell organization in the OSVZ and cortical plate.
Of course, not everything in research is confined to the lab. My daily routine now includes a grueling 1.5-hour commute to the lab and another 1.5 hours back home. While it can be exhausting and sometimes feels like an endless journey, I’ve started using that time to reflect on the day’s findings, brainstorm ideas, or even listen to science podcasts that fuel my curiosity. It’s all part of the experience, and it reminds me just how dedicated I am to unraveling the mysteries of prenatal brain development. But to be honest it’s also really tiring. Coming into the lab 4-5 times a week is fun, but it takes a pretty large physical toll and it’s been taking a lot of willpower to keep pushing myself to get things done.
As I wrap up Week 2, I’m both exhausted and exhilarated. The insights I’ve gathered from those dark room sessions, coupled with my deep dive into gene expression and cannabinoid impacts, have laid the groundwork for what promises to be an exciting next phase of my project. I’m eager to continue my journey with Imaris analysis, and I can’t wait to see what new stories these cells have to tell.
Thank you for sticking with me through these early mornings and busy commutes. Your support means the world, and I’m excited to share more discoveries as I continue further along with my research.
Sources:
https://pmc.ncbi.nlm.nih.gov/articles/PMC4697046/
https://pubmed.ncbi.nlm.nih.gov/31667550/
https://www.nature.com/articles/s41586-024-08351-7
Week 3: Vacation Over, Back to Work!
Hey everyone, I’m back and more energized than ever after an inspiring school trip to Costa Rica! The tropical vibes really recharged my batteries, and now I’m diving headfirst back into my lab work that I can’t wait to share with you.
Returning from Costa Rica felt like hitting a refresh button. The vibrant landscapes and warm culture offered a break from my routine. With a clear mind and a renewed passion, I jumped back into the lab ready to tackle new challenges.
This week, I focused on evaluating some new antibodies that our lab recently acquired. My goal was to determine their viability and quality through immunostaining. The antibodies I worked with include:
SUMO2: Key for post-translational modifications, playing an important role in protein regulation.
CHD3: A powerful chromatin remodeler essential for gene expression during development.
NG2: An exciting marker for neural progenitor cells, offering insights into early brain development.
MCM2: A critical marker for cell proliferation, shedding light on tissue growth dynamics.
I meticulously optimized the staining protocols for each antibody, noting optimal dilutions, staining patterns, and any background signals. Each antibody presented its own set of challenges, and it was incredibly rewarding to witness those "aha!" moments when the staining finally made sense.
Alongside immunostaining, I also immersed myself in tissue culture, a cornerstone of our experimental work. Preparing the culture media is almost like cooking, where every ingredient plays a critical role. Here’s a peek into my process:
Plate Preparation:
I began by coating my culture plates with Poly-D-Lysine (PDL). PDL is essential for enhancing cell adhesion, ensuring that our neural cells stick to the plates properly. I applied the PDL solution to the plates and allowed them to incubate overnight.
2. Media Composition:
The base of my media was DMEM/F12, a nutrient-rich medium ideal for neural cell culture. To this, I added several supplements:
Glutamate: To support neurotransmitter functions and cell metabolism.
Sodium Pyruvate: Serving as an additional energy source.
B27 SDX: A serum-free supplement that provides essential vitamins and antioxidants.
Penicillin/Streptomycin (Pen/Strep): To prevent bacterial contamination.
NR (Nicotinamide Riboside or other relevant nutrient): To support cellular energy metabolism and overall health.
I carefully measured each component, mixed them under sterile conditions, and warmed the media to 37°C before use. This precise blend ensures that our cells receive the optimal environment to thrive.
3. Collecting Samples:
After preparing the media, I collected fresh tissue samples and began setting up the cultures. It’s fascinating how a well-crafted media can mimic the in vivo conditions, allowing cells to maintain their natural behaviors even outside the body.
With the new antibodies validated and tissue cultures established, I moved on to one of my favorite parts of the week, microscopy. Using the Zen software, I conducted detailed imaging on autism-related slides that had been stained for SUMO2, CHD3, NG2, and MCM2. Each slide provided a detailed view into cellular architecture, allowing me to see how these markers were distributed across the tissue. I actually conducted this work to help out another lab group on campus, so it didn’t contribute much to my research aside from giving me some more practice with the microscope.
And yes, I have to mention a lighter moment from this week, the campus cafeteria! It’s not every day you find such a diverse spread of food. From hearty Indian dishes and fresh sushi to classic pizza and crispy fries, the variety is incredible. Even as a vegetarian, I was delighted by the range of options, not to mention the futuristic robot chef that adds a fun twist to lunch breaks!
This week has been a rollercoaster, from fine-tuning new immunostaining protocols and meticulously preparing culture media, every moment has been a step forward in understanding neurodevelopment. Thank you for joining me on this journey of discovery. I can’t wait to share more about my research as I continue my lab journey!
Week 4: Finalizing Experiments and Navigating Life’s Crossroads
Hey everyone, welcome back to another deep dive into my research journey. This week has been all about refining techniques, wrapping up final immunostainings, and balancing the ever-present horror that is college decisions. Let’s break it down.
This week, I put my focus on RNA extraction, specifically using the Qiagen RNeasy kit. I was excited to work with this method because it offers a streamlined and reproducible approach compared to older techniques. Here’s a detailed look at the process I followed:
Sample Lysis: I began by homogenizing my tissue samples in Buffer RLT. This lysis buffer contains guanidine thiocyanate, which is essential for denaturing proteins and inactivating RNases. This step ensures that the RNA is safely released from the cells without being degraded.
Ethanol Addition: Once the samples were fully lysed, I added ethanol. This creates the proper binding conditions for RNA to attach to the silica membrane within the spin column. The precise concentration of ethanol is crucial, it sets the stage for efficient RNA binding and high purity.
Column Binding and Washing: After mixing, I transferred the solution to a spin column and centrifuged it. In this step, RNA binds firmly to the silica membrane, while unwanted components pass through. I then carried out a series of washes using Buffer RW1 and Buffer RPE. These washes remove proteins, salts, and other impurities, ensuring that the final RNA is of high quality.
Elution: Finally, I eluted the RNA with RNase-free water(water without anything that could destroy the RNA). The resulting RNA was pristine and ready for any downstream applications. This method not only minimizes variability but also guarantees reproducibility across samples.
This hands-on experience with the Qiagen kit really highlighted the importance of meticulous protocol adherence in obtaining reliable data. It’s a method that I can see being indispensable for future experiments.
Parallel to my hands-on work with the Qiagen kit, I spent some time diving into the literature on Trizol RNA extraction. I wanted to understand its mechanism and weigh its pros and cons against the Qiagen approach. Here’s what I gathered from my research:
Mechanism of Action:
Trizol is a powerful reagent that uses a combination of phenol and guanidine thiocyanate to both lyse cells and inactivate RNases. Its key strength lies in its ability to denature proteins and separate nucleic acids from other cellular components through phase separation.
Phase Separation Process:
When Trizol is used, the addition of chloroform leads to a clear separation into three phases: an aqueous phase rich in RNA, an interphase containing DNA, and an organic phase with proteins. This separation is particularly effective under acidic conditions, ensuring that RNA remains soluble and isolated.
While Trizol is highly effective and cost-efficient, especially for large-scale experiments, the Qiagen kit offers a more streamlined and user-friendly workflow. The kit’s reliance on silica membrane technology results in more consistent yields and fewer issues with contamination. From my reading (including insights from a MDPI study), I learned that the choice between Trizol and kit-based methods often comes down to the balance between cost, reproducibility, and ease of use. This research has broadened my understanding of RNA extraction and reinforced why I opted for the Qiagen kit for my experiments. It’s all about selecting the right tool for the job.
In tandem with RNA extraction, I finalized the immunostaining of THC-treated slides from gestation weeks 18-24. These slides are the culmination of weeks of practice and represent the final set for my research and microscopy analyses. I meticulously applied the optimized protocols, ensuring consistent staining across all samples. Seeing those distinct, clear staining patterns under the microscope was incredibly rewarding.
I also spent time capturing high-resolution images of GW18 slides using Zen software. These images are vital for my subsequent 3D analysis with Imaris software. However, mounting the slides proved to be a bit of a challenge. Mounting is basically where you put a cover slip on a slide to prevent any damage or contamination later in our work. While I was mounting, my hand kept accidentally hitting the desk because it was too big, making the mounting process a minor acrobatic feat. These little setbacks remind me that even in high-level research, there are always practical challenges to overcome.
Amidst the meticulous lab work, the reality of college decisions has been weighing on me. It’s a stressful time, filled with uncertainty about the future. However, diving into the lab has provided a welcome distraction. The detailed protocols and scientific challenges have not only sharpened my skills but also helped clear my mind, reminding me why I’m so passionate about this field. Sometimes, the best way to handle stress is to focus on the work you love.
This week has been a blend of hands-on technique refinement, in-depth research, and navigating life’s bigger decisions. The experience with the Qiagen RNeasy kit has solidified my understanding of RNA extraction, while my research into Trizol has given me valuable insights into alternative methods. Finalizing immunostaining and capturing detailed images of THC-treated slides are significant milestones that move my project closer to completion.
Thank you for following along on this journey, both the scientific discoveries and the personal challenges. I can’t wait to see what the next chapter holds!
Sources:
https://www.mdpi.com/2305-6304/10/2/48
Additional lab protocols and Qiagen RNeasy kit user manuals.
Week 5: Diving into Data with IMARIS
Hey everyone, welcome back to another deep dive into my research journey! This week, I’ve been transforming raw images into meaningful data by combining high-resolution microscopy with advanced 3D analysis in IMARIS. I captured images across several treatment groups, including methanol (control), fentanyl, THC, and CBD-treated slides, and uncovered some fascinating differences in cellular behavior and marker expression.
My week began once again with microscopy sessions using Zen software. I meticulously documented the cellular landscape on tissue slides under various conditions:
Methanol (Control): These slides served as our baseline, showing the expected cellular architecture and standard marker expression.
Fentanyl: The fentanyl-treated slides revealed notable alterations in cell distribution and changes in marker expression compared to controls.
THC: The THC-treated samples displayed subtle yet intriguing shifts in differentiation markers, suggesting nuanced effects on neural development.
CBD: CBD-treated slides brought their own distinct profile, with differences in both cell counts and marker intensities that point to potential modulatory effects on neurodevelopment.
Each image is a snapshot of the dynamic processes occurring in the tissue, capturing how these substances might be influencing cellular behavior. However, we still have to compare how the methanol treated slides compare to the completely untreated ones. The reason methanol is our control, is that both THC and CBD were both methanol coated, so in order to confirm differences in the sections were caused by THC or CBD and not methanol, we have to use methanol as our control.
Once I had my images, I imported them into IMARIS for detailed 3D reconstruction and quantitative analysis. This powerful software allowed me to:
Visualize Spatial Distributions: I could see the 3D arrangement of cells and markers, which helped in understanding how treatment conditions affect tissue structure.
Quantify Cell and Marker Counts: Detailed plots comparing cell counts and marker intensities across the different treatments were generated, providing a quantitative basis for my observations.
Analyze Significant Differences: Statistical analysis of these plots revealed significant variations between the treatment groups. For instance, fentanyl-treated slides showed a marked shift in cell proliferation patterns, while THC and CBD conditions demonstrated distinct modulations in differentiation marker expression compared to the methanol control.
Here were some of the key differences without any specific spoilers:
Fentanyl vs. Control: The data suggests that fentanyl exposure may significantly alter both the number of cells in key regions and the expression of markers(specifically SOX5) related to differentiation.
THC Slides: THC-treated samples exhibit unique changes that hint at subtle alterations in the developmental trajectory of neural cells.
CBD Slides: The CBD condition, intriguingly, presents a profile that differs from both the control and the other drug treatments, potentially indicating a modulatory role in neurodevelopmental processes.
This week has been a blend of image capture, advanced 3D analysis, and rigorous data interpretation. By examining methanol, fentanyl, THC, and CBD-treated slides side by side, I’m piecing together a clearer picture of how each treatment impacts cellular dynamics and marker expression during development. These insights are a significant step forward in my research, bringing me closer to understanding the molecular mechanisms affected by these substances.
Thank you for joining me on this journey of discovery. Every data point and every image brings new insights, and I can’t wait to see where these findings lead next!
Week 6: Data Analysis, Funding Challenges, and Creative Breakthroughs
Hey everyone, welcome back to another week in the lab! This week has been a whirlwind of advanced analysis, practical lab work, and even a bit of creative fun, all against the backdrop of some important discussions about research funding.
The week kicked off with more detailed analysis using IMARIS. I imported our latest microscopy images, now enriched by protein expression data, and began creating intricate 3D reconstructions. These reconstructions are essential to accurately quantify and visualize the spatial distribution of our markers, allowing me to draw deeper comparisons between our control samples and those treated with marijuana and fentanyl.
In parallel, I turned my focus to protein extraction using a Qiagen BCA kit designed for high-yield recovery of cellular proteins. This method involved a series of precise steps:
Lysis: I began by lysing our samples in a specialized buffer that disrupts cell membranes while preserving protein integrity.
Binding and Washing: Proteins were then captured using a silica-based membrane, followed by a series of washes to remove contaminants.
Elution: Finally, the purified protein was eluted in an appropriate buffer, ready for downstream analysis and correlation with our imaging data.
This extraction process has broadened our dataset, giving us another layer of insight into how substance exposures affect the protein expression in developing tissues.
A significant part of this week was also marked by a lab meeting where we discussed the impact of recent NIH funding cuts. The conversation was both sobering and motivating, reminding us of the challenges in the world of research, but also igniting creative ideas for securing alternative funding streams and optimizing our experimental workflows. It was a crucial reminder that scientific progress often comes with hurdles, and collaboration is key to overcoming them.
In the midst of busy lab work and funding discussions, I also dove into the creative side of science. Using Adobe Illustrator, I meticulously formatted the images for our upcoming poster presentation. This process involved:
Adjusting the visual layout for clarity.
Enhancing image resolution and contrast to showcase our data effectively.
Integrating graphs and statistical plots that summarize our quantitative analyses.
Crafting the poster and slides is an integral part of communicating our findings, and it’s incredibly satisfying to see the story of our research take shape visually.
On a lighter note, I must mention the unexpected highlight of the week, a delightful meeting treat! During one of our lab discussions, I enjoyed a fantastic bagel with jalapeño cream cheese. It was a small moment, but one that provided a welcomed break and a burst of energy amid our intense discussions about funding and data.
This week has been a blend of rigorous data analysis, hands-on protein extraction, strategic planning amid funding challenges, and creative poster design. Each component has contributed to our broader goal of uncovering the intricate effects of prenatal substance exposure on neural development. While the funding news was a sobering reminder of the challenges ahead, the combination of technical advances and creative problem-solving has left me both inspired and ready for the next phase of this journey. Thanks for following along, here’s to another week of pushing the boundaries of our research!
Week 7: From Pixels to Plates
Hey everyone, welcome back! This week I balanced the artistic side of science with some hands‑on cell work. Here’s how it went down:
With our data analyses wrapping up, I dove into Adobe Illustrator to assemble the research poster for our upcoming symposium. First, I imported the 3D reconstructions and quantitative plots from IMARIS, then carefully arranged them alongside concise figure captions. I standardized fonts and colors to ensure consistency, used alignment grids to balance text blocks, and added call‑outs highlighting key findings, like the altered Ki67 and Sox2 distributions in drug‑treated sections. Adjusting contrast and vectorizing certain diagrams made the images pop, and exporting as a high‑res PDF means the final print will look crisp. It’s always satisfying to see raw data transform into a clear visual narrative.
On the bench, I focused on tissue culture work to collect fresh fetal brain tissue for our next set of assays. I started by coating multiple six‑well plates with Poly‑D‑Lysine to promote cell adherence. Meanwhile, I prepared media composed of DMEM/F12 supplemented with glutamate, sodium pyruvate, B27 SDX, Pen/Strep, and NR, warming it to 37 °C before use. Under sterile conditions, I retrieved fresh tissue sections, performed gentle enzymatic digestion with papain to dissociate cells, and then centrifuged and resuspended the pellet in culture media. After counting cells with a hemocytometer, I plated them at optimal density and placed the plates in our incubator. These primary cultures will allow us to observe live‑cell responses to drug treatments and collect additional material for future immunostaining and molecular assays.
It was a week of contrasts, spending mornings perfecting layout details in Illustrator and afternoons nurturing delicate neural cultures at the hood. Seeing the poster come together gave me a real sense of progress, while setting up the cultures reminded me of the patience required for biological work. Next week, I’ll monitor cell viability, change media, and begin preliminary drug exposures in culture. Stay tuned for updates on how these live‑cell assays compare to our fixed‑tissue findings!
Week 8: Polishing Projects and Expanding Perspectives
Hey everyone, welcome back! This week I balanced final design work with deep literature integration and an inspiring campus visit. Here’s how it went down.
I spent the first half of the week perfecting every detail of the immunostaining figures for my symposium poster. In Illustrator, I fine-tuned the fluorescence overlays, rebalancing the Sox2, Ki67, GFAP, and DAPI channels for maximum clarity, added vector-precision scale bars and zone labels, and aligned all panels using guides to ensure a polished, publication-worthy layout. Adjusting color curves and exporting as a high-resolution PDF ensures that the final print will faithfully represent the intricate cellular patterns we’ve documented.
Alongside the visuals, I conducted a meta-analysis of published studies on prenatal marijuana and opioid exposure to contextualize our Bay Area data. By extracting effect sizes, gestational age ranges, and demographic information, I was able to compare our findings, particularly the altered Sox2/Ki67 distributions, with broader trends in the literature. I then added these insights into a PowerPoint narrative that begins with the 19 percent prenatal marijuana use rate in the Bay Area, describes our tissue-section immunostaining and IMARIS 3D analyses, highlights my quantitative contributions, and closes with the public health implications of our work.
To cap off the week, I visited Case Western Reserve University’s campus in Cleveland and was immediately struck by its welcoming atmosphere. Strolling through the residence halls, I saw how thoughtfully designed the living spaces are, with comfortable common rooms and study nooks that make balancing academics and social life effortless. Sampling local favorites in the nearby Uptown district, from cozy coffee shops to eclectic eateries, gave me a taste of the vibrant student dining scene. Conversations with current undergraduates about their favorite campus traditions, club activities, and the wealth of research opportunities taught me that CWRU offers not only top-tier academics but also a tight-knit community where I could grow both personally and intellectually.
And that’s about all for this week everyone, thank you for tuning in!
Week 9: Commitment and Communication
Hey everyone, welcome back! This week has been all about celebrating a huge milestone and sharpening how I share our work with others.
I’m thrilled to share that I have officially committed to Georgetown University! After weighing factors like research opportunities, campus culture, and the strong pre-med program, Georgetown felt like the perfect fit. Accepting their offer was both exhilarating and a relief, and it’s given me a renewed sense of purpose as I head into the final weeks of this project.
With the commitment behind me, I returned to my poster and focused on making the visuals more accessible to anyone, regardless of neuroscience background. I introduced high-contrast color schemes for key markers, and replaced technical axis labels with intuitive icons. I also fine-tuned the legends so that viewers can immediately grasp patterns in cell counts or marker intensities without needing a deep dive into the methods. The result is a poster that still conveys our rigorous data but invites a broader audience to understand the story we’re telling.
Finally, I revisited my PowerPoint to integrate a section on earlier organoid studies that inspired this project. I summarized landmark findings on how brain organoids model early development, explained why organoid research provides critical insights into cellular self-organization, and connected those lessons to our tissue-section work. By tracing this lineage, from organoid breakthroughs to our own imaging and analysis, I’ve created a more compelling narrative that situates our findings within the larger field of developmental neuroscience.
This week’s blend of celebration, design refinement, and contextual framing has left me energized and ready to present our work with clarity and confidence. Thanks for following along, and stay tuned as we head into the final stretch!
Week 10: Wrapping It All Up
Hey everyone!
This week was all about tying everything together, months of effort, analysis, and discoveries finally coming into focus. I spent most of my time finalizing my research poster and polishing my presentation script. Seeing all of my figures, image panels, and graphs laid out in one cohesive visual format was honestly crazy. It’s wild how these scattered data points from early on, just fluorescent blurs and counts, now tell a full story about how marijuana can shift the course of early human brain development. I made a few last-minute tweaks to figure layouts using Adobe Illustrator, like refining the spatial placement of SATB2 and SOX5 comparison images to help viewers more clearly see patterns across zones like the VZ, OSVZ, and CP.
On the presentation side, I practiced giving my talk, tightened transitions between slides, and worked on making the scientific terms more digestible for people who might not have a background in neuroscience. Preparing to share this project publicly made me realize just how much I’ve learned over the past ten weeks, from immunostaining to image analysis to communicating complex science with clarity and confidence.
As a small thank-you to the amazing team that supported me throughout this journey, I brought in a box of pastries for everyone in the lab. It felt good to give back in a small way, especially after all the patience, mentorship, and encouragement they’ve shown me since day one. Honestly, sharing those pastries over some casual lab banter made the end of the project feel even more real and memorable. It’s hard to say goodbye to a space that taught me so much and made me feel like I truly belonged. Thank you to all of you for staying with me on this ten week journey, it’s been really fun!