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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