In my last post, I addressed many of the learning goals which I set for myself at the beginning of the summer. A skill acquisition type of objective, working with the confocal microscope, replaced my initial objective to work with stereology software. As confocal microscopy was deemed more appropriate for the task at hand (to view, label, and quantify types of neurons based on their neurotransmitter type and subregion localization within the ventral tegmental area). I had hoped to master the use of this equipment by the end of my internship. However I have found that mastery of any skill requires far longer than a single summer’s worth of practice. This is one point which truly served as a learning experience regarding the real world of scientific research, and of the working world in general. Along with confocal microscopy, immunohistochemistry was another skill which I had desired to master by the summer’s end. I have come to find that such skills might take a life time to master, and that one can never truly perfect them but instead constantly improves technique with practice. Scientists in this field, including the ones who spent time training me this summer, have spent decades practicing a great variety of laboratory techniques, and they too are constantly learning from colleagues and improving from their own in-success. From this, I learned that each and every skill that can be used in science takes hard work and dedication to reach a level of expertise. There are no short cuts or quick routes to an easy success.

This summer, I did learn a great deal about the basics of immunohistochemistry and confocal microscopy, and I practiced these skills to the point where I have laid a significant layer of groundwork to build off of in my future endeavors in the laboratory. By the end of my time on the job I had a compiled a box full of slides that I had prepared using immunohistochemistry of which I would view under the confocal microscope.

I managed to achieve a level of independence, of which I am quite proud, where I was allowed to, and comfortably did, adjust various steps in the procedures and protocols. My work was always taken seriously, and by the end of the summer, researchers, with years of experience, with whom I worked closely would even consult me about the procedures that I had spent time fine-tuning if they were about to start a new project using a similar technique. The one other undergraduate, aside from myself, who spent the summer as an intern with the same lab group became a great colleague, as she and I would frequently teach other the techniques and skills we had learned from our separate mentors, effectively learning from one another.

In fact, this was something I truly found amazing about the nature of the work experience in a research laboratory environment. There exists such a high level of collaboration amongst lab members that everyone, regardless of experience or age, is treated and treats one another with the utmost respect. Rather than striving to compete with co-workers, this lab experience showed me the extraordinary benefits of working along side and aiding them, so that everyone becomes invested in each other’s success rather achieving success through the failure of those around them.

There are a few words of advice that I would want to give a student interested in pursuing this kind of internship. In my opinion, one of the most essential components of achieving a sense of satisfaction from the internship experience is to ask any and all questions that come to mind. As an intern, a student is there to learn through real-world experience in that field while simultaneously performing a service of some kind to their host organization. In my experience this summer, I found that just about everyone is more than happy to help out the intern. Sometimes the other people working here can even learn a thing or two within their own field of expertise through a thought provoking question. It can: inspire the expert to look at something in a new way, cause the student to appear engaged and intellectually curious about their job, and also give the student-intern an answer to their question. Another piece of advice I would add is remember that nothing is black-and-white with a strict right or wrong answer, not even science. Pretty much everything is open to interpretation, including both new results/findings as well as currently accepted theories. Never be afraid to criticize/question someone else’s opinion(that is, as long as you aren’t ever rude/condescending about it), as such criticisms will typically be appreciated and usually help to build stronger support for the researcher’s argument. One thing to be wary of is the potential to over-criticize. I noticed this summer, during some of the lab meetings which I attended weekly, that some people could get carried away with criticism, perhaps in an attempt to show off or maybe out of spite. Either way, it becomes fairly obvious when someone ceases to criticize for the purpose of improvement/scientific progress and begins to do so for more selfish reasons.

Another important thing to remember as a summer intern is to make friends with coworkers and maintain these relationships. It is a great way to network and you never know who you might end up working with in the future. Having a good relationship with coworkers is also a necessity in laying the foundation for establishing an environment conducive to the high level of collaboration discussed above. Most simply, being friends with your coworkers will make your summer internship far more enjoyable. It is also vital to remember that just as the people you’re working with will typically be more than happy to help you out and answer your questions, it is important for you to be just as willing to help them. Whether it be through a skill that you have been practicing throughout your internship or just through a more broad knowledge base due to being a current student at a university while they might have a more focused yet narrow knowledge base, don’t forget that being a summer intern near the bottom of the totem pole does not mean that you are useless to those that you’re working with.

A truly amazing learning experience for me, this summer internship provided more for me than I could have imagined possible over the extent of a couple months. The skills that I was taught and honed are some which I can apply to many different experiments in my field throughout my future in the world of science. Regarding purely skill based learning, in addition to immunohistochemistry and confocal microscopy, I received certification in live mouse handling. Due to the highly sensitive nature of live animal use in laboratories, this process was extraordinarily thorough requiring rigorous learning, training, and testing. The process eventually resulted in my acquisition of these certificates:

More so than the various skills I learned this summer however, the greatest aspect of my summer learning entails the ability to now apply this experience and the lessons I’ve learned to the working world. This internship also provided me with a much needed experience, in that it provided me with the experience as to what a career in the laboratory science actually entails. Before this summer, my plans for a future career were based upon an amalgamation of ideas that I had assembled within my own mind. Now I have a much more accurate and realistic expectation of just what exactly working in a neuroscience research laboratory really means. My mentor also helped to guide me, beyond the capacity of anyone who has not directly gone through the process, toward taking the best possible steps to following the path of professional neuroscience laboratory research.

As of right now, I still plan on pursuing a career in research but with a newly improved perspective on the actual means of getting there along with the actual nature of the job. In the more short term future, I have just been hired to work at a laboratory position on campus. It is in the slightly different field of cellular and molecular biology which will entail research on a more broad level than just the brain. I will be able to apply the skills and practices that I learned over the summer to this job. During which I hope to learn new skills as well as broadening my own perspective toward the field of research. This is something that my mentor this summer highly stressed, the importance of training under multiple teachers to learn how to think about and approach a problem from more than one way. Another goal which I still plan on pursuing during my time as an undergraduate is the completion of an independent research project. During my time spent at Columbia this summer, I definitely learned a good deal about the pre-experiment process. This entails performing a seemingly excessive amount of research within databases to read as much as possible in all major and many minor publications, so that before the brainstorming process even begins the researcher will have gained a substantial foundation of knowledge from classic experiments to the newest, most recently published data. It is in this way that the scientific community truly flourishes, when scientists build off of the findings of both their contemporaries and historic researchers.

 

Although we have not yet worked on writing lab reports , for they are not written until the end of a given project(which could take years), I have been training and practicing to interpret my findings each day. This entails both an understanding of the underlying principles behind the experimental procedures I am performing, and more importantly, development of my ability to troubleshoot. Troubleshooting is a vital skill in the world of lab science. It involves encountering an error of some sort, and knowing the assortment of possible reasons that could have caused my data to show results in contention with my hypothesis. At which point the next step is to compile a list of potential reasons for this discrepancy.

The first set of possibilities might require looking at possible problems with each individual step in the procedure. It is essential to alter as few steps as possible per each trial of the procedure. This helps to specify exactly what change causes the resulting effect. One such instance might include changing the ratio of a reagent used. During immunohistochemistry, for example, the brain sections I was staining for the presence of tyrosine hydroxylase(TH) and vesicular glutamate transporter 2(vglut2) were demonstrating an oversaturation of magenta (the color which was supposed to indicate the presence of vglut2), so that this color almost entirely masked all others that should have been seen. I resolved this problem by lowering the ratio of the primary antibody raised against vglut2 from 1:400 to 1:500, which caused less non specific staining.

If my problem had not been solved, then I would have needed to take note of such, and either revert my change in the protocol to be used for future attempts at the procedure(if it had made the problem worse) or keep it in the protocol(if it improved the result slightly) and add another change to a different part of the procedure to further improve my results. If no changes successfully result in the product I need, then it may become time to change my hypothesis. This is the scientific process, essentially educated trial and error until results are seen.

I have also found that utilizing a concept that I picked up in a philosophy class at Tulane will greatly assist me in coming up with my own research plans in the future. A model described to me, known as top-down thinking, calls for starting with the broadest, most general parts of an idea and narrowing your plan down at each level of thought, wherein the uppermost three tiers in the proposed model should be the most expansive in scope, and therefore the most well thought out. If one of these upper level parts of the hypothesis are wrong, it could mean starting my project from scratch with a completely new idea. Whereas a mistake in the lower tiers is not quite so devastating, and will require only minor changes to minute details of the procedure. This can be done relatively easily, without an overwhelmingly negative effect on the project as a whole.

At this point in my internship, I am most proud of the level of independence that I have attained while working in the lab. I have begun to feel comfortable enough with various procedures to the point where I make small changes to various aspects of the protocol in order to optimize potential results. Now, on most days, my mentor does not need to instruct me on what to do or on how to do it(but is of course still always available to answer any questions I might have). By giving me a broad overall goal to achieve, my mentor has allowed me to develop and implement my own style of achieving it. Although I have been getting familiar with a few different procedures and techniques, it has become apparent that it is very significant to understand how any specific task, that I or another lab member performs, relates to the larger picture of the project as a whole. Learning about the interconnections of the many aspects of the project, each performed by a different member of the lab, allows me to better understand the methods of a professional researcher in this field so as to maximize potential quality of the results while emphasizing their relevancy to the scientific community and to society as a whole.

I have also begun practicing the skill of finding and utilizing the results of previously published data and papers. This is integral for formulating research plans, as it allows the experimenter to build upon the findings of other scientists, both contemporaries who research similar areas and more historic scientists whose works serve as the foundation for modern science.

Another skill I have been developing is proper use of the confocal microscope. Not the ordinary piece of equipment that can be found in any lab or classroom, the laser scanning confocal microscope is a powerful machine that allows the up-close viewing of fluorescently labeled mouse brain sections. It produces such a highly magnified view that the user can actually see each individual neuron on the brain slice and what type of neurotransmitter that cell fires. This is where the staining from immunohistochemistry comes into play. Having sectioned a mouse brain on a vibratome into slices 3 microns thick, I would then perform their staining using immunohistochemistry (a procedure that takes advantage of the biologically natural system of antibodies to allow selective labeling). Once having stained for the selected targets (in my case: TH and VGLUT2 in the VTA), I take the brain slice, which I have since mounted in a glass slide, and analyze it under the confocal microscope. I have included a picture of a brain slice that I personally sectioned, stained, and analyzed under the microscope.

In the pictures: red indicates tyrosine hydroxylase(TH) which signifies a dopaminergic neuron, blue is indicative of DAPI which binds to all cellular nuclei (so that a blue dot surrounded by red proves that there is a neuron producing dopamine and that the red is not just free floating dopamine), the magenta highlights vesicular glutamate transporter 2(VGLUT2) which signifies the presence of glutamate(wherein the blue dot within magenta indicates a glutamate projecting neuron)

My internship site is Columbia University Medical Campus in New York City. I work as a summer student in both Sulzer and Harrison Laboratories. Dr. Sulzer and Dr. Harrison are both Ph.Ds in their respective fields. Dr. Harrison is in charge of a research project, within the anesthesiology department, as its principal investigator, while Dr. Sulzer is the principal investigator of a project within psychiatry department. For the summer, I am registered as a psychiatry student at Columbia, although multiple fields/departments can technically apply to my position. The principal investigators and their respective lab teams are all currently working together on a conjoined project, wherein each member of either lab leads a specific project within a more broad area of research. The umbrella topic which incorporates all of these research projects entails the multitude of effects and responsibilities that dopamine has within the brain. This overarching subject matter serves as a foundation for a great variety of possible diseases/disorders to tackle. To illustrate the magnitude of this subject matter, medical problems from which we stand solutions by studying dopamine projections throughout the brain include: Parkinson’s (among other motor function disabilities), addiction, and schizophrenia. On a more day to day level, dopaminergic neurons and their projection targets influence just about every decision a person makes in a day. The specific project which I am a part of focuses on the mechanisms of addiction, especially in regard to alcohol, by utilizing animal models. It is fairly well accepted throughout the scientific community that drugs of abuse exert their powerful grip on the brain by “hijacking” the rewards circuitry (a process of reward/consequence that provided humans a vast evolutionary advantage throughout the span of our species existence). This project is headed by an alumni of Tulane University who is currently a Ph.D student at Columbia. The goal of her project is to demonstrate the existence and heterogeneity of dopamine neuron sub populations in the ventral tegmental area (VTA) based on their projection targets throughout the brain. My role will incorporate two main tasks. First, I will assist in the labeling of neurons, within mice which have been exposed to ethanol(alcohol), via immunohistochemistry. Particularly we will be using retrobead labeling to analyze the various targets of VTA neurons, which will fluorescently highlight the dopamine neurons in the VTA and the specific brain structure that each neuronal population fires to. I will be responsible for applying the antibodies and staining which actually make these neurons and retrobeads visible under the microscope. The neurons will appear as four possible colors, where in: blue(DAPI stain) will highlight all neurons in the brain section, green(retrobeads) will show the neurons in the target brain structure, red (Nissl stain) will show the dopaminergic neurons within that VTA that project to targets brain structure neurons, and yellow will show other types of neurons (e.g. GABA, glutamate, etc…). The brain regions of interest targeted by the VTA include: the medial Prefrontal Cortex, nucleus Accumbens(nAC) lateral shell, nAC medial shell, nAC core, and the basolateral amygdala. A two day process, I have already prepared my first few slides with the slices of a single mouse brain. This leads me to my next duty which will be to quantify the subpopulations of dopamine neurons in the VTA based on their projection target. I will be using a confocal microscope which allows a clear, highly magnified view of these neurons and their targets. I am in the process of being trained on this microscope which is a highly delicate and complex machine. As part of my training, I have looked at the previously mentioned slides which I had prepared, and I was able to visually see exactly what I had been working on earlier that week. I have also attended two lab meetings, which are held on Friday mornings. During which the members of the lab discuss the work that they have done since the last meeting, so that each person involved in the lab contributes information from their specific project, providing the whole team with a more full perspective on the overall project.

-Jason