1. Supervising Faculty Member: Dr. Flynn
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

2. We maintain a normal amount of sodium and water-
Vasopressin = anti diuretic hormone

3. 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

4. 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
70% of VP neurons express NK3 receptor
NK3R

5. NK3Rs play a role in the regulation of the release of the hormone vasopressin.
Injections of a selective NK3R antagonist (SB ) 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.

6. 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.

7. Nuclear NK3R What is the physiological function of 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.

8. 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).

9. Method-
Rats 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.

10. Results
Control
2 M Salt loaded

11. 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

12. NK3R associates with transcriptionally active chromatin

13. 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

14. Thanks!
Dr. Flynn
Don Pratt
Dane Jensen
Xihui Xu
Zhaojie Zhang