2022 Art From the Benchtop Exhibit, Selected Works

CONFOCAL MICROSCOPY

Imaged here is Suml49PT, which is a cell line derived for triple-negative breast cancer (TNBC), stained for Aquaporin 3 with Alexafluor 564 and phalloidin is stained with Alexa fluor 488. We allowed Sum l 49PT cells to attach to a chamber slide overnight and then used an immunofluorescence assay to stain them. It was then imaged with a confocal microscope under 60x objective lens and oil. The tissue was incubated overnight with the primary antibodies and stained with the appropriate secondary antibodies the next day. We are measuring the localization for Aquaporin 3 in breast cancer. Aquaporin 3 is associated with the transfer of water and glycerol that has been shown to be associated with cell migration and growth. When I first started taking images of this slide, I was drawn to the spindling from the phalloidin, and was intrigued and entranced by the shapes formed. Under direction of my mentor, I showed the image to people outside of the medical field and they immediately were scared of the image. They described feeling watched and uneasy from it. My submission title reflects the terror they felt from the image without even knowing what it was.

Ms. Powrozek is a laboratory research assistant working with the mentorship of Neelam Sharma-Walia, PhD, in the Center for Cancer Cell Biology, Immunology and Infection.

IMMUNOFLUORESCENCE

The main aim of this experiment was to evidence the specific neuronal types in the bed nucleus of the stria terminalis (BNST) that express the oxytocin receptors. We are looking at a brain slice from a transgenic rat that expresses oxytocin receptors, designed and bred by a laboratory we are in collaboration with in Germany. The brain slice contains the BNST, a brain region involved in the modulation of fear and anxiety. Although the oxytocin expression is not seen here, we observe a specific type of neuron of the BNST that expresses protein kinase C delta (PKCẟ).

The PKCẟ neurons were revealed by immunofluorescence. This technique consists of using first a primary antibody that will recognize and bind specifically PKCẟ neurons. In a second step, we use a secondary antibody that has a fluorescent molecule which recognizes and binds only the primary antibody. The image was taken using a fluorescent microscope at 10x magnification.

I chose to begin with PKCẟ neurons, because they are abundant in the BNST. I processed these brains and tried to troubleshoot the immunofluorescence, because I was not able to visualize PKCẟ neurons. After several attempts, I was advised to do a specific treatment to the tissue called antigen retrieval before immunofluorescence. This treatment helped to visualize PKCẟ neurons in these transgenic rats and allowed me to move forward to the next set of experiments. It is the image we see here.

The image is the result of a new project and international collaboration in my lab, and the common troubleshooting in science we experience to obtain good quality of data. For these reasons, I combined the image of the BNST slice containing PKCẟ neurons with the woman wearing a mask and working in her laboratory during the pandemic. I want to represent the women in science whose hard work and contributions are not recognized as valuable as they should be. They still contributed to the advancement of technology and innovation. Globally, a small percentage of researchers are women, they are awarded less funding than men, and they are less likely to reach high positions in academia. During the COVID-19 global pandemic, women in scientific research experienced more disruption in their work, but they still contributed to the advancement of technology and innovation.

Ms. Olivera-Pasilo is a fifth-year SGPS student working with the mentorship of Joanna Dabrowska, PhD, in the Center for Neurobiology of Stress Resilience and Psychiatric Disorders.

FLUORESCENT MICROSCOPY.

This is a rat brain section used for tracing neurons. The red and green dots are fluorescent retrograde beads that were injected into the brain. The line of green beads follows the path of the needle used for the injection. The red beads are lining a ventricle, a fluid filled cavity in the brain, due to a misplaced injection. The blue dots are the nuclei of all neurons in the section, which have been stained with DAPI (4′,6-diamidino-2-phenylindole). The image was taken at 4x magnification under a fluorescent microscope. This image was produced by a mistake — a misplaced injection caused the red beads to line a ventricle near the injection site of the green beads. This mistake created a "happy little accident,” as Bob Ross would say, and the resulting image took my breath away under the microscope.

For this study, we were tracing the origin of cells in the medial amygdala. To do this, we injected fluorescent retrograde beads into the brain that are designed to travel backwards along the axons of neurons to their source. In this case, we injected red fluorescent beads into the ventromedial hypothalamus (VMH), a brain region generally involved in social approach, and we injected green beads into the bed nucleus of the stria terminalis (BNST), a brain region generally implicated in stress and anxiety. We then took sections of the medial amygdala (MeA), a brain region involved in social behaviors, and counted the number of cells with red or green beads. Cells with red beads would represent MeA neurons that project to the VMH, and cells with green beads would represent MeA neurons that project to the BNST. By doing this, we could count the proportion of MeA cells that project to each region.

Ms. Ritger is a third-year combined MD/PhD student working with the mentorship of Amiel Rosenkranz, PhD, in the Center for Neurobiology of Stress Resilience and Psychiatric Disorders. Funding for their research was provided by the NIH.

IMMUNOFLUORESCENCE

This image shows a brain slice from a rat with a viral injection to express a protein that fluoresces when certain cells are active. The virus is tagged with a green fluorescent protein, so neurons appearing green in the image are expressing the virus. The blue dots represent all the neurons present in the sections, and have been stained with DAPI (4′,6-diamidino-2-phenylindole), a nuclear stain. The image was taken at 10x magnification under a fluorescent microscope. The wispy green lines are axons of neurons that were cut off in a cross section. They reminded me of the way falling snow looks in headlights.

Fiber photometry is a technique that allows you to record brain activity in an experimental animal while the animal is actively engaging in a behavior. This is done by injecting a virus into the brain that fluoresces when neurons are active and implanting a fiber optic cannula into the brain that can measure the emitted light. In our study, the virus was injected into the bed nucleus of the stria terminalis (BNST), a brain region generally implicated in stress and anxiety. This particular virus is designed to travel backwards along the axon of the neurons to their source. In our case, we implanted a fiber optic cannula into the medial amygdala (MeA), a brain region involved in social behaviors, so that we could measure neuronal activity selectively in MeA neurons that project to the BNST. In this way, we measured the activity of MeA-BNST neurons to see if they were more active during social behavior in rats that had been previously exposed to stress.

This approach is a recently developed way to look at brain activity in experimental animals while the animal is activelyengaged in a behavior, which allows for a more meaningful interpretation of neuronal activity. To me, this piece represents the achievement of learning to use a relatively new and advanced technique in neuroscience. I am proud to be a small part of the next generation of neuroscience experiments.

Ms. Ritger is a third-year combined MD/PhD student working with the mentorship of Amiel Rosenkranz, PhD, in the Center for Neurobiology of Stress Resilience and Psychiatric Disorders. Funding for their research was provided by the NIH.

IMMUNOHISTOCHEMISTRY

This confocal image shows a brain slice of a wild-type mouse, focusing on the hippocampus. lmmunohistochemistry was performed, and the stain used is of a lysosome marker — LAMP 1, which is pseudo-colored light blue. The pseudo-colored red stain is V0al — a vacuolar ATPase pump marker located on acidic vesicles within the neuron. Overall, it represents the localization of V0al on lysosomes within the CAI region of the hippocampus. The localization and function of these proton pumps are altered in Alzheimer's disease.

Ms. Mustaly is a seventh-year SGPS student working with the mentorship of Grace Stutzman, PhD, in the Center for Neurodegenerative Disease and Therapeutics.

IMMUNOHISTOCHEMISTRY

Immunohistochemistry was used to examine the level of accumulation of the storage material in the cerebellum. We stained and imaged a 50 μm sagittal brain slice from a mouse model of CLN3 Batten disease and used a 1 OX objective on a widefleld microscope to acquire the image. This study aimed to quantify the amount of storage material (green) and compare to levels in wild-type mice. Additionally, we also used an antibody (glial fibrillary acidic protein, GFAP) to examine astrocytes (red), which can indicate neuroinflammation. We can then test a potential therapy and determine if we can reduce these levels. CLN 3 Batten disease is a fatal neurodegenerative disease in children. A histopathologic feature of the disease is the accelerated accumulation of storage material in the brain. Additionally, one clinical manifestation of the disease is a deficit in motor coordination. The cerebellum is one brain region involved in movement.

Dr. Centa is a postdoctoral research associate working with the mentorship of Michelle Hastings, PhD, in the Center for Genetic Diseases.

IMMUNOHISTOCHEMISTRY

This image is from a 50 μm section of a kidney from a 1.5-month-old mouse. We are working on CLN3 Batten disease, a lysosomal storage disorder with an onset between the ages of 4-10. Symptoms include vision loss, seizures, loss of motor function, behavioral changes and cognitive decline. This disease is caused by mutations in the gene CLN3, which encodes a lysosomal protein, CLN3. Much is still not known about CLN3, including the function, structure and expression. We have generated a genetically modified mouse model to aid in learning more about this disease. To fully characterize this model, we have started examining the expression of CLN3 in various tissue, including the kidney, seen here from our preliminary assessment. The DNA is stained with Hoechst in blue, and our protein of interest, CLN3, in red. A l0x objective was used with a widefleld fluorescent microscope to acquire the image.

Dr. Centa is a postdoctoral research associate working with the mentorship of Michelle Hastings, PhD, in the Center for Genetic Diseases.

FLUORESCENT MICROSCOPY

This is a brain slice from a CRF-Cre transgenic rat. The rat received an injection of a Cre-dependent virus into the central amygdala. The virus then gets expressed in the CRF (corticotropin releasing factor) neurons, and since it contains a fluorescent tag, we're able to visualize these cells without additional staining.

The section was imaged on a fluorescent microscope under a I Ox objective with a 561-nm fluorescent laser to visualize the mCherry tag on the virus. We were using this method to quantify the number of CRF neurons that get transfected with the virus we injected. Our objective was to compare this transfection to another virus. However, we came across limitations of the other virus that prevented us from using it. Thus, we no longer needed to quantify the number of cells transfected by this virus.

I was drawn to this image with the dense cluster of cells and processes. The original photo reminded me of a swarming beehive, which led me to image it with the black and yellow color combination. From there, I started to see the shape of a wasp in the cells, so I overlaid the outline of a wasp and hand-traced the image into its final shape.

I think this experiment can highlight the trial and error that commonly occurs in research. Not every experiment works, but we are still able to learn from our losses and appreciate the beauty along the way.

Ms. Chudoba is a fourth-year SGPS student working with the mentorship of Joanna Dabrowska, PhD, in the Center for Neurobiology of Stress Resilience and Psychiatric Disorders. Funding for their research was provided by NIMHROI.