Michael McNeil and Dr. Jeffrey Edwards Physiology and Developmental Biology
Endocannabinoids are molecules that play an important role in neuromodulation and are also known to be a factor in many peripheral nervous system functions such as appetite and pain sensation. Recent discoveries have also shown that endocannabinoids and their cell-signaling mechanisms are found within the central nervous system, specifically within the hippocampus, an active component of declarative or explicit memory, yet their role in the CNS is largely unknown. Within the hippocampus there are two main categories of neurons, the pyramidal cells and the interneurons, and both are critical for hippocampal function. However, the interneurons appear to be involved in the modulation of neuronal firing. Therefore we hypothesized that these endocannibinoid signaling mechanisms would be found and expressed within the interneurons of the hippocampus.
In order to confirm this hypothesis, we are studying the hippocampus through two methods. First by using a quantitative analysis of the mRNA expression through a reaction known as qRT-PCR. The second assay is a qualitative analysis known as immunocytochemistry (ICC), which stains proteins of interest. The results from the quantitative and qualitative analyses will help us further understand the mechanisms of synaptic plasticity within the hippocampal interneurons and their role in learning and memory.
The first assay done was the qRT-PCR in which we isolated over 50 hippocampal interneurons found within the stratum radiatum from brain slices of rats between 16-28 days old. After applying negative pressure, cells were extracted from the slice and then aspirated into an electrode. Each cell was then placed immediately into a chilled reverse transcriptase reaction mix and run on a BioRad C1000 thermal cycler within two hours to convert cellular mRNA to cDNA. Following a multiplex run with all 16 primer sets at low concentration, the subsequent cDNA was amplified with forward and reverse primers along with the appropriate probe on a BioRad CFX96 qPCR machine set to detect the desired. GAD65 and GAD67 genes were used to identify and confirm that the neurons were indeed hippocampal interneurons, along with other genes to identify different interneuron subtypes. Several other genes are being quantified to confirm expression of several endocannabinoid signaling mechanisms such as TRPV-1, an endocannabinoid receptor, 12-LO, a gene that produces 12-HPETE an endocannabinoid, and NAPE-PLD a protein that is involved in anandamide synthesis, another endocannabinoid.
The data for these interneurons is currently being quantified and analyzed to confirm if there truly is a correlation between hippocampal interneurons and the endocannabinoid signaling mechanisms, but the data up to this point seems promising. We have found multiple endocannabinoid signaling components within the hippocampal interneurons while performing the RT-qPCR. Specifically those cells that expressed either GAD65 or GAD67 (confirming they are interneurons) also expressed various levels of CB1, an endocannabinoid receptors, along with 12-LO and NAPE-PLD, both involved in endocannabinoid synthesis. Therefore we are not only seeing the receptors but also the producers of these molecules.
We are currently using ICC to qualitatively analyze the location of these expressed genes as proteins within the cell. This is done by taking a hippocampal slice, and adding a primary antibody to the tissue slice which will bind to the antigen of interest. A secondary antibody with a fluorescent tag will be added and will bind specifically to the primary antibody. When looking under a confocal microscope we will be able to recognize the location of the protein that was bound by the primary antibody due to the flourescence. The use of this assay will help us determine protein expression of endocannabinoid components within thecells, supporting our mRNA findings within these cells. Depending on our qRT-PCR results we will select several proteins that are being co-expressed in the same cells consistently and perform ICC to confirm their co-expression at the protein level.
There is still some work that needs to be done with calibrating the antibodies so that they work with the particular tissue samples that we use for ICC so little experimental data has been collected, however we are confident that the results will only further confirm are data from the RT-qPCR.
Overall this project was incredibly rewarding. It was challenging at times and it took us much longer than we first proposed. It took longer than we first believed to gain the techniques and skills necessary to perform the particular assays as well as make sure that we have all of the controls with which we can compare our experimental samples. Even with these challenges I learned an incomprehensible amount on the importance of patience, perseverance, and the power of critical thinking while solving different challenges. Most importantly in helped me learn how to be creative on projects and understand exactly what should be done to confirm a hypothesis. There is an incredible amount of preparation that is required for different projects, but also the creativity necessary when challenges and new insights occur. I am grateful for the opportunity to have worked on this project and have had this preparation for the future.