This summer I am working with Dr. Clabough to identify a number of proteins that are vital to brain and neuronal development and may be affected by ethanol exposure. We are using a Fetal Alcohol Spectrum Disorders (FASD) mouse model and have chosen a paradigm that mimics a human third-trimester binge drinking session. Many FASD mouse models demonstrate neuronal deficits that persist into adulthood, though the specific actions of ethanol on the development of the nervous system are still not well understood, especially immediately after the ethanol exposure.
We have isolated protein from FASD and control mouse brains. We are using Western Blotting techniques to isolate specific proteins, such as Huntingtin, Brain Derived Neurotrophic Factor (BDNF), RE1-Silencing Transcription factor (REST), also known as Neuron-Restrictive Silencer Factor (NRSF), and others. We are using densitometry on ImageJ software to determine the relative concentrations of these specific proteins in important brain areas, such as the striatum and cortex.
We are hoping to find changes in the expression of these genes 24 hours after ethanol exposure, and possibly a molecular correlate to a changed striatal neuronal branching that we have also observed at this early time point. The branching of these neurons is of major concern so these neurons connect to transmit signals properly. Therefore, a change in neuronal branching may translate into a similar change in behavioral, motor, or overall cognitive function that can be seen in live models.
I have learned much about basic protein extraction and procedures by running Western Blots and interpreting results. The most important thing I have learned from this summer, is that Westerns rarely work. However, the problem solving skills I have learned from this have made me a better biologist and student in general. I cannot express my gratitude to both Dr. Clabough for all that she has taught me, but also to the Honors Council for allowing me to complete this research.
This summer I have had the privilege to join Dr. Clabough and a collaboration of people working on a project called Turtle Sense, which involves placing sensors in turtle nest to monitor when turtle hatchlings will emerge. The physical placement of an egg-shaped sensor into a turtle nest enables the relay of motion data in the nest back to a communication tower and then to a database. The motion data can then be used to predict baby turtle emergence.
Turtle Sense data from previous years have shown that these sensors are capable of predicting when sea turtles will actually emerge from their nest within a few days. Our method of data collection can divide the motion of a sea turtle nest from day 0-60 into four phases. Phase A consist of a quiet incubation period (roughly 50 days). Phase B is a transition period that usually has some large swings with a frequency of 2-4 swings per a day. This phase ends with a quiet and low motion reading, which is often lower than anything in the preceding weeks. Phase C is the hatching activity, which is typically 4 days long and characterized by erratic, frequent motion. Phase D is a quiet period indicating emergence is imminent.
This summer I’ve been looking at past years’ sensor data collected at Cape Hatteras to better formulate an experimentally outlined procedure for optimizing the way data is collected, as well as how the data can be used to best create a way for other people to implement the same technology in sea turtle nests in other geographic locations. We’ve seen in some data from past years that the graphs can detect non-turtle motion periods at certain points, which may indicate predation from ghost crabs, washouts from the ocean, wind manipulating the sensors, and storms.
We are working to implement a defined protocol for what to do when a nest is found, as well as specifically how each sensor should be placed into a nest to ensure less experimental error. During full nesting season, we can expect well over 200 sea turtle nests. Nest management currently requires blocking off the nest upon discovery, and after approximately 1.5 months, a larger beach enclosure is placed around the nest that essentially blocks off people from crossing that section of the beach until the nest has clearly emerged.
The best part about this research experience is that I actually get to live in Hatteras, NC for the summer. Living down here and interacting with the locals has taught me a lot about how people view sea turtles and their nesting, as well as the differing views that people have over how the beaches should be utilized regarding sea turtle nests. The nature of the project is definitely one that has proved to be a learning experience— we no longer have control over what we’re working with because we’re actually working with live animals that follow their own life cycle.
We know that ethanol exposure during development has many negative consequences for the offspring. This summer I’ve been working with Dr. Erin Clabough investigating mouse brain morphology following a one-time ethanol intoxication event in the early postnatal period for mice (which is equivalent to the third trimester of a human pregnancy). We have harvested mouse brains, followed a Golgi impregnation procedure, sectioned brains using a cryostat, followed staining procedures, used microscopes and computer programs to identify and trace medium spiny neurons, and will in the future analyze the number and types of dendritic spines.
Jamie Ingersoll ’18 undertaking a Golgi staining process on mouse brain sections. The Golgi stain impregnates approximately 10% of neurons with a black pigment. We lost some usable brain sections into the wash, but in most sections, we were able to see the outline of individual neurons under the scope.
In prior study done in Dr. Clabough’s lab, we found increased branching in individual neurons in the striatum of the mouse brains immediately following a developmental ethanol exposure—which is the opposite of what we thought we’d find. We are now investigating different stages of mouse development to see when this branching phenotype disappears.
The most difficult parts of research so far have been when we were sectioning on the cryostat and it fought us for what seemed like years never wanting to yield a usable section and also the staining process where we had to watch as brain after brain slid off the slides into the unusable wash left behind.
Some of our 873 brain tissue sections drying over several days prior to Golgi staining.
This experience has showed me a whole new level of patience and also how important it is to not rush the process—for example, when something like the cryostat decides it will finally cooperate, then prepare to stay for a while and crank because the next day it may not be so kind. But the best part is when everything works and we are able to see and interpret results.
We will present our research at the 2016 Society for Neuroscience meeting in San Diego in November. We hope that our research will provide insights into not just how just one binge of ethanol can affect neurons in the brain, but also how medium spiny neurons are involved in the response to ethanol and how that response may change throughout development.