ANAT 322 Lecture Notes - Lecture 12: Solitary Tract, Dorsomedial Hypothalamic Nucleus, Wild Type
7 Jun 2018
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ANAT 322 Winter 2017
Lectures
Lecture 12:
2. Hypothalamus and the brainstem control energy homeostasis whereas the VTA controls the hedonic
system.
3. POMC neurons are anorexigenic whereas NPY neurons are orexigenic.
Knocking out the leptin receptor in the population of neurons leads to mild obesity which is not the
same as knocking out the populations of POMC neurons themselves that does lead to obesity. Knocking
out the MC3/4R population of neurons in the paraventricular nucleus (PVN) also leads to obesity as well
as knocking out the leptin gene.
Scientists thought that if you knock out NPY or AgRP mice would become lean by the same logic. This
is not the case as nothing seemed to happen to body weight but these are potent orexigenic peptides.
When they ablated the neuronal population of NPY/AgRP, mice indeed stopped eating and died from
starvation.
4. Mice are insensitive to diphtheria toxin because they do not express the receptor.
Researchers artificially expressed the receptor in those neurons by putting a gene for the receptor
under control of the AgRP gene so once the transgenic mice expressed the DTR only in that population
of neurons, they treated the mice with diphtheria toxin which bound to the receptor causing the
neurons to die.
5. Immunohistochemistry with antibody against AgRP showed that after the mice were treated with
diphtheria toxin the population of AgRP neurons decreased. They used in situ hybridization to double
check and there are no neurons when they used a probe against AgRP after injection of DT.
The body weight of these mice was looked at after single DT injection and it showed that those mice
expressing DTR had a decrease in body weight.
7. When they removed the population of neurons then mice were not interested in eating anymore and
because knock outs of NPY or AgRP were not involved in this phenotype, they thought that there might
be something else in that population of neurons that is causing the effects. They thought that maybe
gamma-aminobutyric acid (GABA) expressed in these neurons was responsible for the phenotype.
8. They used DTR mice and they used a GABA receptor agonist in an osmotic mini-pump which is put
subcutaneously under the skin of mice. Once inserted into the skin, they start to pump because of
differences in osmotic pressure in the pump compared to the surrounding but the pumping rate can be
controlled by the researcher.
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ANAT 322 Winter 2017
Lectures
9. There were two groups of mice that were DTR expressing mice or transgenic mice and they were
treated with DT (double injection) but only one of these mice got the pump with the GABA agonist.
Food intake drops after DT injection in both groups of mice but those treated with the GABA agonist
(mimics GABA), after several days, they go back to the control levels so there was rescued of the
phenotype which supported their hypothesis.
11. The ventricular cavities are filled with CSF which flows only in one direction and the flow is slow.
They used this to understand the brain region to the phenotype. If you have a drug and put it in the
lateral ventricle and there is an effect then the drug maybe interacting with an area close to either of
the ventricles but if you put the drug in the fourth ventricle and we observe an effect, then the areas
involved can only be close to the fourth ventricle.
12. They injected the GABA agonist into either the third or the fourth ventricle.
13. The bottom curve shows mice treated with DT without GABA agonist. If we focus on the upper two
curves we can see that for the top curve they put the drug into the fourth ventricle whereas for the third
curve they put the drug in the third ventricle.
Putting the drug into the fourth ventricle is more potently restoring feeding behavior. If you put the
same dose into the third ventricle, it will eventually reach the fourth ventricle where the site of action is
but the drug will be diluted which is why we see a smaller effect. If we put the drug directly into the
fourth ventricle, the concentration of the drug will be higher and thus an area around the fourth
ventricle is responsible for feeding behavior.
14. They did an experiment in which they injected mice with the drug but used a marker for neuronal
activation called Fos which is known to be upregulated in neurons activated. GABA is an inhibitory
neurotransmitter so when it is around and reaching the target cell, it inhibits the cells and there is
downregulation of Fos.
They looked for differential expression of Fos through different regions of the brain and they
compared mice treated with the drug and the control mice (vehicle). The biggest difference they found
in terms of downregulation of the Fos signal was in the parabrachial nucleus (PBT) but they also saw
some differences in the nucleus of the solitary tract (NTS), both regions located in the brainstem. In the
dorsomedial hypothalamus (DMH) they did not see any difference.
15. They then injected the drug directly into the brain areas (PBN and NTS), not into the ventricles, and
only injections in the PBN result in the mice increasing feeding behaviors (top curve). Injections in the
NTS did not resulted in mice increasing eating (bottom curve).
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Document Summary
Lecture 12: hypothalamus and the brainstem control energy homeostasis whereas the vta controls the hedonic system, pomc neurons are anorexigenic whereas npy neurons are orexigenic. Knocking out the leptin receptor in the population of neurons leads to mild obesity which is not the same as knocking out the populations of pomc neurons themselves that does lead to obesity. Knocking out the mc3/4r population of neurons in the paraventricular nucleus (pvn) also leads to obesity as well as knocking out the leptin gene. Scientists thought that if you knock out npy or agrp mice would become lean by the same logic. This is not the case as nothing seemed to happen to body weight but these are potent orexigenic peptides. When they ablated the neuronal population of npy/agrp, mice indeed stopped eating and died from starvation: mice are insensitive to diphtheria toxin because they do not express the receptor.
