In the unusually warm summer of 2008, I was lucky enough to arrange a vacation studentship with Dr Glenda Gillies and Dr Simon McArthur at Imperial College. The project I was taking part in, would investigate whether neonatal Lipopolysaccharide (LPS) treatment, which simulates an inflammatory stress, would have a long-term impact in the brain, specifically on the cytoarchitecture of the adult midbrain dopaminergic (DA) neurons of the ventral tegmental area (VTA) and substantia nigra (SNc), which are highly sensitive to environmental changes.
As a Neuroscience student, I had spent a considerable period of time in laboratories as part of my degree studies, but never on a project of this length or depth. It therefore gave me the opportunity to become immersed in a particular topic and learn novel techniques. This was not the only aspect of the project that was novel; the division had recently relocated to a brand new, state-of-the-art facility, so I was to be working in fairly luxurious surroundings.
The project took advantage of a study at Kings College London run by Dr Kevin O'Byrne, looking into neonatal LPS treatment on the reproductive axis, specifically in adult female rats, leaving the brains of the male rats available for this project. Before any immunohistochemistry could take place, the perfused brains had to be sliced and this involved using a cryostat - a machine I would become very well accustomed to. The first stage required a quick crash course in rat neuroanatomy to identify the start of the SNc, so that as I was slicing rostral to caudal, I would know when to start and finish collecting brain sections. After nearly 16 brains, I quickly realised the dramatic effect of even minute adjustments, to the cryostat stage and ones technique; mastering the brushing skills for the collection of the 10Î¼m thick slices.
The second stage involved immunohistochemically marking the DA neurons with an antibody raised against tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis, and a selective marker of midbrain DA cells. This particular part of the experiment allowed me to appreciate the need for accuracy and care especially when dealing with biological tissue like brain sections. The third stage involved placing the brain sections with labelled cells on slides, which again required a certain level of dexterity with brushes. These where then dried in increasing percentages of alcohol and fixed with a cover slip and xylene.
Finally with a light microscope, photos of the VTA and SNc where taken and then counted in ImageJ - which seemed like a blessing after I had been informed by a number of researchers that they had previously had to look down the microscope with a clicker for long hours in order to count. Indeed this stage of the study demonstrated to me how vital a virtue patience is in scientific research but it is also when I felt most excited and determined to obtain data and determine what they meant. In the end, as with many research studies, the results suggested some answers, but raised as many, if not more, questions - something which has been in itself an interesting lesson to learn!
Importantly, the study allowed me to improve my proficiency and confidence in the laboratory exponentially before I embark on my Dissertation in the final year of my degree, and I truly became absorbed in the subject matter, to the extent that on returning to University I selected to take a course on glucocorticoids and programming. Moreover it was an invaluable insight and opportunity to be in an active and friendly research environment, for which I am indebted to the BSN.
Shervin Poladi, University of Edinburgh