Characterization and Tracing of Thermoregulatory Neurons: Introduction

Since spring semester of my freshman year, I have been working in Dr. John Griffin’s lab.  Dr.  Griffin is a neuroscience professor at the college, and his research focuses on determining the thermoregulatory pathways in the hypothalamus.  In general, the lab studies the effects that certain drugs have on the firing rate of neurons to determine what receptors and molecules might be at play in thermoregulation.  Lately, however, I have been working on two new projects that could provide major breakthroughs in the study of thermoregulation.

The first project that I am working on is one that will hopefully allow us to easily trace axonal pathways in the brain.  For several years, Dr. Griffin has been developing a method that utilizes gold nanoprobes to visualize axonal connections.  The method uses miniscule gold particles that are conjugated with two separate antibodies that allow them to be taken up into presynaptic neurons and then to diffuse up to the neurons’ somas.  By injecting the nanoprobes into a certain area of the brain, we can determine what other areas send axons (and thus information) to that area based on where the nanoprobes travel. This new method is extremely important because it is the only method that allows for axonal tracing in live tissue.

Unfortunately, we have had some issues with visualizing the nanoprobes because they tend to be very dim and are difficult to differentiate under a microscope.  Over the last few months, I have been working with Dr. Griffin in an effort to create a better way to visualize the nanoprobes.  Ultimately, we decided that the best method would be to use a secondary antibody stain to attach a fluorescent antibody to the nanoprobes after they have diffused throughout the cells.  Once we decided this, I formed a protocol and ordered all of the necessary materials.  I have tried the new protocol twice and the results look promising, but there is definitely still some fine tuning that needs to be done.  Hopefully, I will be able to work all of the kinks out of the protocol so that we can more easily trace the axonal pathways of the brain.

The other project I will be working on this summer is introducing fluorescence in situ hybridization (FISH) into the lab.  FISH is an extremely powerful tool that allows researchers to determine whether specific cells are transcribing a specific mRNA sequence (and thus producing a particular protein).  At first, I will simply be using FISH to study the efficacy of the nanoprobes.  By studying whether labeled neurons are producing the protein that the nanoprobes are binding to or not, I will be able to determine whether the nanoprobes are actually binding specifically to the protein of interest or not.  This project will allow us to fine tune FISH for our lab and will provide us with the basic knowledge that we will need to further pursue FISH in the lab.  Once I have a basic grasp of FISH, I will attempt to design a protocol in which I use FISH to determine the differences between the classes of thermoregulatory neurons.  Eventually, I hope to be able to characterize thermoregulatory neurons simply based on the proteins they produce.