Current-clamp in whole-cell patch clamping is apparently more difficult than other types of electrophysiological recordings such as voltage-clamp because cells are less stable.
Moreover, POMC neurons in the ARC are more difficult to record from for their size (?) and other reasons.
And recordings from the ARC are difficult in general because of high movement of the tissue because it is positioned close to the third ventricle.
Finally, due to variable and low degree of co-expression of appetite-regulating peptides and serotonin receptors, the current project I'm working on takes that much longer.
SOCS-3 a better marker of activation and inhibition by leptin than c-Fos?
The one (one of a few) disadvantage of c-Fos as a marker of recent neuronal activity is that it only shows neurons activated by a certain response in question and not those inhibited by the said response.
One solution -- at least in terms of neuronal activation in response to leptin -- could be SOCS-3.
SOCS-3
- is a suppressor of cytokine signaling-3
- ''an intracellular protein induced by activation of cytokine receptors such as the leptin signaling by blocking phosphorylation of the receptor and downstream STAT proteins involved in leptin receptor-activated signal transduction [45,46].'' (Baskin et al., 2000)
- ''In CHO cells transfected with ObRb, expression of SOCS-3 inhibits leptin-induced tyrosine phosphorylation of JAK2 proteins, suggesting that SOCS-3 is a leptin-regulated inhibitor of proximal leptin signaling in vivo [47].'' (Baskin et al., 2000)
So if we use c-Fos as a marker of a response to leptin, only neurons that are activated by leptin (such as ARC POMC neurons) will be shown (Elias et al., 1999). But is we use SOCS-3 as a marker of a response to leptin, we could identify neurons that are activated by leptin (ARC POMC neurons) AND those that are inhibited by leptin (such as ARC NPY/AgRP neurons) (Elias et al., 1999).
references:
- Baskin DG et al. (2000). SOCS-3 expression in leptin-sensitive neurons of the hypothalamus of fed and fasted rats. Regulatory Peptides;92:9-15.
- Elias CF, Aschkenasi C, Lee C, Kelly J, Ahima RS, Bjorbaek C et al. (1999). Leptin differentially regulates NPY and POMC neurons projecting to the lateral hypothalamic area. Neuron;23:775–86.
One solution -- at least in terms of neuronal activation in response to leptin -- could be SOCS-3.
SOCS-3
- is a suppressor of cytokine signaling-3
- ''an intracellular protein induced by activation of cytokine receptors such as the leptin signaling by blocking phosphorylation of the receptor and downstream STAT proteins involved in leptin receptor-activated signal transduction [45,46].'' (Baskin et al., 2000)
- ''In CHO cells transfected with ObRb, expression of SOCS-3 inhibits leptin-induced tyrosine phosphorylation of JAK2 proteins, suggesting that SOCS-3 is a leptin-regulated inhibitor of proximal leptin signaling in vivo [47].'' (Baskin et al., 2000)
So if we use c-Fos as a marker of a response to leptin, only neurons that are activated by leptin (such as ARC POMC neurons) will be shown (Elias et al., 1999). But is we use SOCS-3 as a marker of a response to leptin, we could identify neurons that are activated by leptin (ARC POMC neurons) AND those that are inhibited by leptin (such as ARC NPY/AgRP neurons) (Elias et al., 1999).
references:
- Baskin DG et al. (2000). SOCS-3 expression in leptin-sensitive neurons of the hypothalamus of fed and fasted rats. Regulatory Peptides;92:9-15.
- Elias CF, Aschkenasi C, Lee C, Kelly J, Ahima RS, Bjorbaek C et al. (1999). Leptin differentially regulates NPY and POMC neurons projecting to the lateral hypothalamic area. Neuron;23:775–86.
The co-expression of POMC, NPY and 5-HTRs in the ARC
Have you ever been wondering about what the are the co-expression patterns of pro-opiomelanocortin (POMC) and the neuropeptide Y (NPY) neurons in the arcuate nucleus of the hypothalamus (ARC) with what serotonin receptors (5-HTRs)? Yes, I thought so... Here is little info for you!
I have included the methods used to label the peptide (to label a protein: immunireactivity = IR; to label protein's mRNA: in situ hybridisation = ISH) and the animal model with the validation of the model where relevant.
POMC & 5-HT2CRs
wt RAT
50% to 80% of POMC cells (POMC-IR) (from rostral to caudal ARC respectively) express 2Cs (2C-ISH) in a wt rat (Heisler et al. 2002)
POMC-tau/lacZ+/- MOUSE
75% of POMC cells (beta-galactosidase-IR) express 5-HT2CRs (5-HT2CR-IR - but uses a dodgy antibody) in a POMC-tau/lacZ+/- (Lam et al., 2008)
28% of 5-HT2CR cells (5-HT2CR-IR - but uses a dodgy antibody) express POMCs (beta-galactosidase-IR) in a POMC-tau/lacZ+/- (Lam et al. 2008)
validation:
100% of beta-galactosidase-IR express POMC-ISH in a POMC-tau/lacZ+/- (Lam et al., 2008)
NPY & 5-HT1BRs
NPY-GFP MOUSE
16.5% (1/6) of NPY cells (GFP-IR) express 5-HT1BRs (5-HT1BR-ISH) in a NPY-GFP mouse (Heisler et al., 2006)
33% (1/3) of 5-HT1BR cells (5-HT1BR-ISH) express NPY (GFP-IR) in a NPY-GFP mouse (Heisler et al., 2006)
validation:
99.5% of GFP-IR express AgRP-ISH in a NPY-GFP mouse (Heisler et al., 2006)
Of note, apparently there are differences in expression of POMC in mice and in rats (Cowley et al., 2001) so that might explain the differences (in addition to the different genetic model).
I have included the methods used to label the peptide (to label a protein: immunireactivity = IR; to label protein's mRNA: in situ hybridisation = ISH) and the animal model with the validation of the model where relevant.
POMC & 5-HT2CRs
wt RAT
50% to 80% of POMC cells (POMC-IR) (from rostral to caudal ARC respectively) express 2Cs (2C-ISH) in a wt rat (Heisler et al. 2002)
POMC-tau/lacZ+/- MOUSE
75% of POMC cells (beta-galactosidase-IR) express 5-HT2CRs (5-HT2CR-IR - but uses a dodgy antibody) in a POMC-tau/lacZ+/- (Lam et al., 2008)
28% of 5-HT2CR cells (5-HT2CR-IR - but uses a dodgy antibody) express POMCs (beta-galactosidase-IR) in a POMC-tau/lacZ+/- (Lam et al. 2008)
validation:
100% of beta-galactosidase-IR express POMC-ISH in a POMC-tau/lacZ+/- (Lam et al., 2008)
NPY & 5-HT1BRs
NPY-GFP MOUSE
16.5% (1/6) of NPY cells (GFP-IR) express 5-HT1BRs (5-HT1BR-ISH) in a NPY-GFP mouse (Heisler et al., 2006)
33% (1/3) of 5-HT1BR cells (5-HT1BR-ISH) express NPY (GFP-IR) in a NPY-GFP mouse (Heisler et al., 2006)
validation:
99.5% of GFP-IR express AgRP-ISH in a NPY-GFP mouse (Heisler et al., 2006)
Of note, apparently there are differences in expression of POMC in mice and in rats (Cowley et al., 2001) so that might explain the differences (in addition to the different genetic model).
The ARC is full of neurons other than POMC/CART and AgRP/NPY
I have long suspected that the neurons of the arcuate nucleus of the hypothalamus (ARC) express much more then the anorexigenic POMC/CART and the orexigenic AgRP/NPY peptides. And in the last two days I learned what else can be found in the ARC. It's quite a selection...
POMC and CART... mostly co-expressed, essential for energy balance regulation
AgRP and NPY... co-expressed, essential for energy balance regulation
kisspeptin... assumed to connect reproduction to energy balance
neurokinin B
dynorphin... apparently the last three are co-expressed together within one neuron
dopamine
SOC3... co-expressed with both POMC and AgRP, associated with energy balance
TMEM18... found generally in neurons, associated with energy balance
and the list continues. It makes it very interesting to study this nucleus and the complex neural networks.
POMC and CART... mostly co-expressed, essential for energy balance regulation
AgRP and NPY... co-expressed, essential for energy balance regulation
kisspeptin... assumed to connect reproduction to energy balance
neurokinin B
dynorphin... apparently the last three are co-expressed together within one neuron
dopamine
SOC3... co-expressed with both POMC and AgRP, associated with energy balance
TMEM18... found generally in neurons, associated with energy balance
and the list continues. It makes it very interesting to study this nucleus and the complex neural networks.
There are 4 classes of potassium membrane channels
Who would have thought that I owe wikipedia for today's 'Wow!' moment that there are 4 major classes of potassium membrane channels.
Significance?
This is such a wonderful finding because my learning about resting and action membrane potentials makes much more sense. More specifically, I know that two different classes of potassium channels are responsible for the resting and for the action membrane potential, which is something I suspected and now have in writing :)
So the resting membrane potential of a typical neuron is negative (close to -60mV) because the membrane is more permeable to potassium ions at rest via the tandem pore domain potassium channels. Therefore, at rest, the positive potassium ions leave the neurons down the electrochemical gradient making the inside of the neurons more negative.
Of note, if the membrane of the resting neuron was more permeable to sodium ions than to potassium ions, the resting membrane potential would be much more positive (e.g. +40mV) because the positive sodium ions would enter the neuron down their electrochemical gradient.
On the other hand, when an action potential arrives, the membrane becomes relatively more permeable to sodium than to potassium ions via opening of voltage-gated sodium channels. As then the positive sodium ions enter the neurons, making the membrane potential more positive (e.g. +40mV).
When the membrane (action) potential reaches a treshold voltage (e.g. +40mV), the membrane again becomes relatively more permeable to potassium ions and less to sodium ions. Now the voltage-gated potassium channels open and allow the positive potassium ions out of the neuron making the membrane potential more negative again.
In the refractory period the membrane is more permeable to potassium ions and the ion concentrations inside and outside of the cell normalise back to the baseline of the resting membrane potential.
Well, I am stil learning so I suspect the above is still too basic at best and plain wrong at worst.
Significance?
This is such a wonderful finding because my learning about resting and action membrane potentials makes much more sense. More specifically, I know that two different classes of potassium channels are responsible for the resting and for the action membrane potential, which is something I suspected and now have in writing :)
So the resting membrane potential of a typical neuron is negative (close to -60mV) because the membrane is more permeable to potassium ions at rest via the tandem pore domain potassium channels. Therefore, at rest, the positive potassium ions leave the neurons down the electrochemical gradient making the inside of the neurons more negative.
Of note, if the membrane of the resting neuron was more permeable to sodium ions than to potassium ions, the resting membrane potential would be much more positive (e.g. +40mV) because the positive sodium ions would enter the neuron down their electrochemical gradient.
On the other hand, when an action potential arrives, the membrane becomes relatively more permeable to sodium than to potassium ions via opening of voltage-gated sodium channels. As then the positive sodium ions enter the neurons, making the membrane potential more positive (e.g. +40mV).
When the membrane (action) potential reaches a treshold voltage (e.g. +40mV), the membrane again becomes relatively more permeable to potassium ions and less to sodium ions. Now the voltage-gated potassium channels open and allow the positive potassium ions out of the neuron making the membrane potential more negative again.
In the refractory period the membrane is more permeable to potassium ions and the ion concentrations inside and outside of the cell normalise back to the baseline of the resting membrane potential.
Well, I am stil learning so I suspect the above is still too basic at best and plain wrong at worst.
Membrane capacitance to measure vesicular release
My notes are based on a very old paper by Angelson & Betz (1997). I am not sure how much relevance it still has today. So keep that in mind.
Membrane capacitance
- capacitance of a membrane can be used to measure exocytosis/endocytosis, vesicular release and neurotransmitter release
- because as vesicles fuse with the membrane, its area and volume increase and it is able to 'store' more charge (explained here)
- however, this can be done well only with vesicles larger than 50 nm in diameter so apparently not that many...
- also, for this technique whole-cell clamp is less useful than perforated-cell clamp and than on-cell clamp
Membrane capacitance
- capacitance of a membrane can be used to measure exocytosis/endocytosis, vesicular release and neurotransmitter release
- because as vesicles fuse with the membrane, its area and volume increase and it is able to 'store' more charge (explained here)
- however, this can be done well only with vesicles larger than 50 nm in diameter so apparently not that many...
- also, for this technique whole-cell clamp is less useful than perforated-cell clamp and than on-cell clamp
A method of finding unknown proteins of a specific function
...by creating an artificial protein of this specific function of interest.
I know it is not so much related to appetite regulation but I still think it's cool!
Details
In cellular biology, scientists can design an artificial protein structure. Then they can go and look into 100,000s let's say yeast colonies and see what mutant cells can't exist without this protein structure. When they find these mutants, saved by the protein, they can see what the mutant gene/protein is and go on and fully characterise it.
So simply by designing an artificial protein structure of a known function, endogenous proteins of this same functionality can be find and characterised.
Reference: http://www.sciencemag.org/content/325/5939/477.abstract Kornmann et al., 2009. An ER-Mitochondria Tethering Complex Revealed by a Synthetic Biology Screen. Science, 325(5939), 477-481.
I know it is not so much related to appetite regulation but I still think it's cool!
Details
In cellular biology, scientists can design an artificial protein structure. Then they can go and look into 100,000s let's say yeast colonies and see what mutant cells can't exist without this protein structure. When they find these mutants, saved by the protein, they can see what the mutant gene/protein is and go on and fully characterise it.
So simply by designing an artificial protein structure of a known function, endogenous proteins of this same functionality can be find and characterised.
Reference: http://www.sciencemag.org/content/325/5939/477.abstract Kornmann et al., 2009. An ER-Mitochondria Tethering Complex Revealed by a Synthetic Biology Screen. Science, 325(5939), 477-481.
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