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The role of tachykinin neurokinin 3 receptor within the nuclei of neurons in the paraventricular nucleus of the hypothalamus and its properties of translocation Sean Bell University of Wyoming Neuroscience Program Supervising Faculty Member: Dr. Flynn We maintain a normal amount of sodium and water- Vasopressin = anti diuretic hormone VP is an essential hormone and is synthesized in neurons in the PVN and SON- and is released into the blood. VP prevents dehydration- key factor in the regulation of body water How are VP neurons turned on or activated? • Magnocellular VP neurons express a wide range of receptors. NK3R exists throughout the nervous system especially within the paraventricular nucleus. VP NK3R 70% of VP neurons express NK3 receptor NK3Rs play a role in the regulation of the release of the hormone vasopressin. • Injections of a selective NK3R antagonist (SB222200) into the brain prevents the release of VP in response to hyperosmolarity (due to a salt load). • NK3Rs that are expressed on VP neurons are activated and internalized to the cytoplasm in response to acute hyperosmolarity. Neurokinin Receptors: • So hyperosmolarity causes the release of a ligand (neurokinin B) in the PVN that then binds to its receptor and is internalized to the cytoplasm. This is to be expected. • But, then the NK3R (a plasma membrane receptor) appears in the cell nucleus- this shouldn’t happen. Nuclear NK3R • Therefore, NK3Rs are activated by injection of an NK3R agonist or osmotic challenge (intragastric load of hypertonic saline). • Interestingly, once NK3R has been activated, NK3R has been found not only within the cytoplasm but also within the nucleus. • What is the physiological function of nuclear NK3R? • My project sought to identify the subnuclear compartment that associates with NK3R. One likely subnuclear compartment and target of nuclear NK3R is Chromatin • • Chromatin is abundant in the nucleoplasm and is a complex of DNA and proteins (histones). Histone proteins are integral to chromatin and play an important role in controlling gene expression. • Hypothesis: Nuclear NK3R associates with histone (we used histone H4 as a marker of chromatin). MethodRats were given an (IG) load of 2 M NaCl (6ml). Control rats were given no load. The PVN was then isolated and processed for electron microscopy. Immuno-electron microscopy- antibodies against H4 or NK3R. Each antibody was visualized by administering a secondary antibody attached to either a 6 nm or 15 nm gold bead. Analysis: Images captured at 40,000 x and stitched together – 42 nuclei analyzed per group/condition. The area of each nucleus was determined along with the total number of NK3Rs, the percentage of co-localization with histones, and the percentage of NK3Rs found in pairs. Results Control 2 M Salt loaded Summary • NK3R associates with chromatin• Chromatin can be active or silent• Acetylation of H4 indicates regions of gene transcription • Experiment repeated but with antibodies against acetylated H4 NK3R associates with transcriptionally active chromatin Conclusion • NK3R is trafficked into the nucleus, in vivo. • My data show for the first time an association of NK3R with chromatin, specifically with H4 and acetylated H4. • We hypothesize that NK3R may play a role in gene expression. On going experiments are exploring this. • This identifies a novel mechanism by which a membrane receptor may affect the genomics of the neuron Thanks! Dr. Flynn Don Pratt Dane Jensen Xihui Xu Zhaojie Zhang