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.
How is the brain important for the regulation of appetite?
We know that maintaining energy balance is essential for keeping a healthy weight i.e. for not developing obesity with all its negative consequences on our life expectancy and mental and physical health.
We also know that brain is a key organ which regulates the energy balance.
But what evidence do we have that convinces us that it is specifically the brain that is the key regulator of energy balance?
There are many lines of evidence which establish the brain, in particular the hypothalamus, as the key regulator of energy balance. However, among the most convincing examples are probably genetic studies of some of the most common monogenic causes of human obesity.
These studies link specific proteins to brain circuits responsible for the regulation of energy balance, specifically to the hypothalamic leptin–melanocortin signalling pathway (see the figure).
In this pathway, leptin, which is secreted from the adipose tissue in proportion to the size of the fat deposits, stimulates leptin receptors on proopiomelanocortin (POMC)-expressing neurons in the arcuate nucleus of the hypothalamus (ARC), a key site of central regulation of energy balance. Subsequently, prohormone convertase 1 (PC1) cleaves the POMC protein into α and β melanocyte stimulating hormones (MSHs). MSHs stimulate melanocortin 4 receptors (MC4Rs), expressed exclusively in the brain, to ultimately reduce food intake and increase energy expenditure.
Deficiency in these components of the pathway – namely leptin, leptin receptor (LepR), pro-opiomelanocortin (POMC), prohormone convertase 1 (PC1), melanocyte stimulating hormone (MSH) and melanocortin 4 receptor (MC4R) – result in positive energy balance and consequently in obesity (Montague et al., 1997; Clement et al., 1998; Krude et al., 1998; Jackson et al., 1997; Yeo et al., 1998).
Figure legend: Most common human monogenetic causes of obesity are linked to the brain, supporting the role ot the brain in the regulation of energy balance. Protein deficiencies resulting in human monogenetic obesity are highlighted in red (adapted from Oswald and Yeo, 2007).
We also know that brain is a key organ which regulates the energy balance.
But what evidence do we have that convinces us that it is specifically the brain that is the key regulator of energy balance?
There are many lines of evidence which establish the brain, in particular the hypothalamus, as the key regulator of energy balance. However, among the most convincing examples are probably genetic studies of some of the most common monogenic causes of human obesity.
These studies link specific proteins to brain circuits responsible for the regulation of energy balance, specifically to the hypothalamic leptin–melanocortin signalling pathway (see the figure).
In this pathway, leptin, which is secreted from the adipose tissue in proportion to the size of the fat deposits, stimulates leptin receptors on proopiomelanocortin (POMC)-expressing neurons in the arcuate nucleus of the hypothalamus (ARC), a key site of central regulation of energy balance. Subsequently, prohormone convertase 1 (PC1) cleaves the POMC protein into α and β melanocyte stimulating hormones (MSHs). MSHs stimulate melanocortin 4 receptors (MC4Rs), expressed exclusively in the brain, to ultimately reduce food intake and increase energy expenditure.
Deficiency in these components of the pathway – namely leptin, leptin receptor (LepR), pro-opiomelanocortin (POMC), prohormone convertase 1 (PC1), melanocyte stimulating hormone (MSH) and melanocortin 4 receptor (MC4R) – result in positive energy balance and consequently in obesity (Montague et al., 1997; Clement et al., 1998; Krude et al., 1998; Jackson et al., 1997; Yeo et al., 1998).
Figure legend: Most common human monogenetic causes of obesity are linked to the brain, supporting the role ot the brain in the regulation of energy balance. Protein deficiencies resulting in human monogenetic obesity are highlighted in red (adapted from Oswald and Yeo, 2007).
Electrophysiology for dummies alias what is fast capacitance, slow capacitance and R-series?
As I promised yesterday, some basic electrophysiology knowledge is coming next as I'm currently learning this technique.
Slow capacitance (C-slow)
- can be viewed as the storage of charge in a cellular (in this case neuronal) membrane
- by pressing the ''Auto'' button in PatchMaster, it is subtracted from the recording for a more accurate result
- it is based on the size of the cell, therefore, on the size of the cell's membrane
- therefore, we can't do much about slow capacitance (can't decrease it)
- therefore, scientists focus on decreasing the fast capacitance to improve the recordings
Fast capacitance (C-fast)
- can be viewed as the storage of charge in a pipette (which is used to patch on cells)
- likewise, by pressing the ''Auto'' button in PatchMaster, it is subtracted from the recording for a more accurate result
- can be improved (decreased) by changing the properties of the pipette (e.g. coating the pipette)
R-series
= series resistance
- therefore measured in ohms
- can be viewed as resistance to the flow of current which is measured in the cell and in the contents of the pipette (their contents mix during whole cell patching and that's why we fill the pipette with an intracellular solution, i.e. one that mimicks the contents of the cell)
- on part of the pipette, the flow is blocked by the pipette's diameter; and on part of the cell, the flow is blocked by the contents of the cell such as the nucleus and other organelles
- we try to have the resistance of the tip and the cell as low as possible for ideal recordings
Slow capacitance (C-slow)
- can be viewed as the storage of charge in a cellular (in this case neuronal) membrane
- by pressing the ''Auto'' button in PatchMaster, it is subtracted from the recording for a more accurate result
- it is based on the size of the cell, therefore, on the size of the cell's membrane
- therefore, we can't do much about slow capacitance (can't decrease it)
- therefore, scientists focus on decreasing the fast capacitance to improve the recordings
Fast capacitance (C-fast)
- can be viewed as the storage of charge in a pipette (which is used to patch on cells)
- likewise, by pressing the ''Auto'' button in PatchMaster, it is subtracted from the recording for a more accurate result
- can be improved (decreased) by changing the properties of the pipette (e.g. coating the pipette)
R-series
= series resistance
- therefore measured in ohms
- can be viewed as resistance to the flow of current which is measured in the cell and in the contents of the pipette (their contents mix during whole cell patching and that's why we fill the pipette with an intracellular solution, i.e. one that mimicks the contents of the cell)
- on part of the pipette, the flow is blocked by the pipette's diameter; and on part of the cell, the flow is blocked by the contents of the cell such as the nucleus and other organelles
- we try to have the resistance of the tip and the cell as low as possible for ideal recordings
c-Fos as a marker of neuronal activation - pluses & minuses
Yes, I know, c-Fos for the third time! But bear with me because a new exciting topic of electrophysiology will be coming shortly as I'll be learning that technique.
c-Fos or other markers used in our lab?
In our lab, we haven't used any other markers of neuronal activation (despite researching this alternative last year). Other markers crop up in papers from time to time but c-Fos is best established and most widely used in our field and therefore, works best for our purposes.
Still, what are the pluses & minuses of using c-Fos as a marker of neuronal activity?
+ c-Fos marks recently active neurons
+ can be used together with physiology research and immunohistochemistry techniques
+ is best established and most widely used in our field so we can compare our results with other people's results
- it isn't a direct marker of neuronal activity
- some neurons may be acitvated but c-Fos doesn't have to be transcribed and translated i.e. c-Fos isn't in a pathway for all stimuli/responses
- time course of c-Fos expression might differ cell-to-cell and response-to-response so it needs to be established individually for each experiment
c-Fos or other markers used in our lab?
In our lab, we haven't used any other markers of neuronal activation (despite researching this alternative last year). Other markers crop up in papers from time to time but c-Fos is best established and most widely used in our field and therefore, works best for our purposes.
Still, what are the pluses & minuses of using c-Fos as a marker of neuronal activity?
+ c-Fos marks recently active neurons
+ can be used together with physiology research and immunohistochemistry techniques
+ is best established and most widely used in our field so we can compare our results with other people's results
- it isn't a direct marker of neuronal activity
- some neurons may be acitvated but c-Fos doesn't have to be transcribed and translated i.e. c-Fos isn't in a pathway for all stimuli/responses
- time course of c-Fos expression might differ cell-to-cell and response-to-response so it needs to be established individually for each experiment
Neurons in the brain are like stars in the universe
At least for me and at least in this youtube video :)
http://www.youtube.com/watch?v=90cj4NX87Yk&feature=related
Also, I like the part where the animation shows the exchange of the ions.
http://www.youtube.com/watch?v=90cj4NX87Yk&feature=related
Also, I like the part where the animation shows the exchange of the ions.
c-Fos peak in the history & alternative markers of neuronal activation
history of c-Fos as a marker of neuronal activation?
In the scientific literature, c-Fos was for the first time referred to as a marker of neuronal activation in:
1. Dragunow, M. & Faull, R. (1989). The use of c-fos as a metabolic marker in neuronal pathway tracing. Journal of Neurosci Methods, 29(3), 261-265. (paid)
2. Hoffman, G.E., Smith, M.S. & Verbalis, J.G. (1993). c-Fos and Related Immediate Early Gene Products as Markers of Activity in Neuroendocrine Systems. Frontiers in Neuroendocrinology, 14(3), 173-213.
at least as far as I could find in a brief literature search.
alternative markers of neuronal activation?
1. c-Jun
2. pERK
3. NGFI-B
= nerve growth factor IB
- ''the expression of NGFI-B after both routes of administration is indicative of cellular activation within the pPVN in parallel with secretion of ACTH.'' (Ogilvie et al., 1998)
It seems that selecting the best marker of neuronal activation depends on the neuronal population and the response to a particular stimulus studied.
In the scientific literature, c-Fos was for the first time referred to as a marker of neuronal activation in:
1. Dragunow, M. & Faull, R. (1989). The use of c-fos as a metabolic marker in neuronal pathway tracing. Journal of Neurosci Methods, 29(3), 261-265. (paid)
2. Hoffman, G.E., Smith, M.S. & Verbalis, J.G. (1993). c-Fos and Related Immediate Early Gene Products as Markers of Activity in Neuroendocrine Systems. Frontiers in Neuroendocrinology, 14(3), 173-213.
at least as far as I could find in a brief literature search.
alternative markers of neuronal activation?
1. c-Jun
2. pERK
3. NGFI-B
= nerve growth factor IB
- ''the expression of NGFI-B after both routes of administration is indicative of cellular activation within the pPVN in parallel with secretion of ACTH.'' (Ogilvie et al., 1998)
It seems that selecting the best marker of neuronal activation depends on the neuronal population and the response to a particular stimulus studied.
c-Fos (also cFos, cfos)
c-Fos (also cFos, cfos)
what it is?
- is a member of immediate early gene family, which contains transcription factors
how it works?
- because cFos is an 'immediate early gene' transcription factor, it is itself made (=transcribed & translated into a protein) 'immediately early' when cell is stimulated/activated by almost any stimulus; transcription factor then means that cFos helps to transcribe (alone or with other proteins) other genes into proteins, which are required by the cell when the cell is activated in response to the particular stimulus
comparison to?
- to a Bible? it is created/written very early on and helps others to get created/converted later on
also of interest?
- it is a proto-oncogene/protein, i.e. a gene/protein which may become an oncogene/protein; proto-oncogenes code for protein which are involved in a cell cycle, cell growth and differentiation
use?
- in neuroscience: used as an indirect marker of neuronal activation because cFos mRNA or cFos protein are upregulated when cell/neuron fires action potentials and marks recent neuronal activity
Would you have a better comparison for the role of c-Fos as a marker of neuronal activity? If yes, please let me know in the comments.
what it is?
- is a member of immediate early gene family, which contains transcription factors
how it works?
- because cFos is an 'immediate early gene' transcription factor, it is itself made (=transcribed & translated into a protein) 'immediately early' when cell is stimulated/activated by almost any stimulus; transcription factor then means that cFos helps to transcribe (alone or with other proteins) other genes into proteins, which are required by the cell when the cell is activated in response to the particular stimulus
comparison to?
- to a Bible? it is created/written very early on and helps others to get created/converted later on
also of interest?
- it is a proto-oncogene/protein, i.e. a gene/protein which may become an oncogene/protein; proto-oncogenes code for protein which are involved in a cell cycle, cell growth and differentiation
use?
- in neuroscience: used as an indirect marker of neuronal activation because cFos mRNA or cFos protein are upregulated when cell/neuron fires action potentials and marks recent neuronal activity
Would you have a better comparison for the role of c-Fos as a marker of neuronal activity? If yes, please let me know in the comments.
The parabrachial nucleus (BPN) is directly or indirectly affected by serotonin
Or in other words that the parabrachial nucleus (BPN) is directly or indirectly affected by serotonin (5-HT) coming from some of the nine serotonin-producing nuclei in the brain.
Off-topic: Of note, serotonin is also produced in the gastrointestinal tract (vast majority of it in the human body in fact) but it doesn't cross the blood brain barrier. And as a result, it doesn't concern us greatly in the brain (though peripheral - non-brain - serotonin may still affect the brain via affarent sensory neuronal pathways).
Details?
By this I mean that the PBN most likely expresses both serotonin 1B and 2C receptors (5-HT1BRs and 5-HT2CRs).
The 5-HT1BR mRNA expression is detected in PBN by in-situ hybridisation histochemistry (ISHH) techniques (Bruinvels et al., 1993). cFos is a member of an immediate early gene family of transcription factors. As such it responds quickly and transiently to a vast range of neuronal stimuli. This allows us to use is as a marker of neuronal activation. Co come back to presence of 5-HT1BR in the BPN, more research shows that administration of a selective 5-HT1BR agonist such as CP-94253 induces cFos-immunoreactivity in the PBN (Lee et al., 1998). This further supports presence of functional 5-HT1BRs in the PBN.
Similarly to the 5-HT1BR, 5-HT2CR mRNA expression is detected in many brain regions including the PBN (Molineaux et al., 1989; Mengod et al., 1990), though the literature is not specific about the specific subdivisions of the nucleus; we know that the PBN is traditionally divided into medial and lateral: the formed composed of medial PBN 'proper' and lateral PBN, the latter composed of external, internal, waist, ventral, dorsal, superior and central. We also know that d-fenfluramine is a serotonin releaser and serotonin re-uptake inhibitor but also that it has affinity for all three serotonin 2 receptor subtypes in the order of 2C > 2B > 2A, i.e. with highest affinity for 5-HT2CR (Knight et al., 2004). Furthermore, d-fenfluramine induces cFos-IR in rat lateral and dorsal PBN. From that we can infer that 5-HT2CR is expressed mostly in the lateral and dorsal subdivisions of the lateral PBN.
This was the long way to support the claim that the PBN most likely expresses both 5-HT1BRs and 5-HT2CRs. We also know that the PBN receives axonal projections from the dorsal raphae (Petrov et al., 1992), which synthetise and secrete serotonin. Therefore, we can propose that the PBN may be directly stimulated by serotonin.
As for the indirect stimulation of the PBN by serotonin, we need to delve into the model of serotonin-mediated anorexia via two distinct populations of neurons in the arcuate nucleus of the hypothalamus (ARC). In the ARC, serotonin activates Gq-coupled 5-HT2CRs on proopriomelanocortin(POMC)-expressing neurons and inhibits Gi-coupled 5-HT1BRs on agouti-related peptide(AgRP)-expressing neurons. The product of POMC, alpha-melanocyte stimulating hormone (alpha-MSH), and AgRP are endogenous ligands of melanocortin 4 receptors (MC4Rs); the former activates and the latter inhibits MC4R-expressing neurons. Therefore, upon serotonin stimulation of the two populations of neurons, the release of alpha-MSH and AgRP can be altered, leading to increased likelihood of activation of MC4R-expressing neurons. This ultimately results in the serotonin-mediated anorexia, i.e. reduction of food intake. We know that MC4Rs are essential (though not necessarily the only) for the effect of serotonin on food intake because the serotonin-induced anorexia is abolished in MC4R-knockout mice as it is for that matter in 5-HT1BR- and 5-HT2CR-knockout mice.
We also know that MC4Rs are expressed widely throughout the central nervous system (CNS) and among other sites also in the PBN (Kishi et al., 2003; Liu et al., 2003; Mountjoy et al., 1994). Furthermore, we know that the ARC projects directly to the PBN (Wu et al., 2008; Bromberger et al., 1998; Moga et al., 1990a&b; Sim et al., 1991). From that we can imply that the PBN can be also indirectly affected by serotonin via the ARC 5-HT1BR- and 5-HT2CR-expressing AgRP and POMC neurons.
Significance?
From previous research, we already know that in the brain serotonin stimulates 5-HT2CRs on POMC neurons in the ARC, which project to the paraventricular nucleus of the hypothalamus (PVH), where they stimulate MC4R-expressing neurons to ultimately reduce food intake and cause weight reduction. But the above things that I learned today have brought the PBN much more into the picture for me, expanding the complexity of the neural pathways that control appetite and body weight.
Off-topic: Of note, serotonin is also produced in the gastrointestinal tract (vast majority of it in the human body in fact) but it doesn't cross the blood brain barrier. And as a result, it doesn't concern us greatly in the brain (though peripheral - non-brain - serotonin may still affect the brain via affarent sensory neuronal pathways).
Details?
By this I mean that the PBN most likely expresses both serotonin 1B and 2C receptors (5-HT1BRs and 5-HT2CRs).
The 5-HT1BR mRNA expression is detected in PBN by in-situ hybridisation histochemistry (ISHH) techniques (Bruinvels et al., 1993). cFos is a member of an immediate early gene family of transcription factors. As such it responds quickly and transiently to a vast range of neuronal stimuli. This allows us to use is as a marker of neuronal activation. Co come back to presence of 5-HT1BR in the BPN, more research shows that administration of a selective 5-HT1BR agonist such as CP-94253 induces cFos-immunoreactivity in the PBN (Lee et al., 1998). This further supports presence of functional 5-HT1BRs in the PBN.
Similarly to the 5-HT1BR, 5-HT2CR mRNA expression is detected in many brain regions including the PBN (Molineaux et al., 1989; Mengod et al., 1990), though the literature is not specific about the specific subdivisions of the nucleus; we know that the PBN is traditionally divided into medial and lateral: the formed composed of medial PBN 'proper' and lateral PBN, the latter composed of external, internal, waist, ventral, dorsal, superior and central. We also know that d-fenfluramine is a serotonin releaser and serotonin re-uptake inhibitor but also that it has affinity for all three serotonin 2 receptor subtypes in the order of 2C > 2B > 2A, i.e. with highest affinity for 5-HT2CR (Knight et al., 2004). Furthermore, d-fenfluramine induces cFos-IR in rat lateral and dorsal PBN. From that we can infer that 5-HT2CR is expressed mostly in the lateral and dorsal subdivisions of the lateral PBN.
This was the long way to support the claim that the PBN most likely expresses both 5-HT1BRs and 5-HT2CRs. We also know that the PBN receives axonal projections from the dorsal raphae (Petrov et al., 1992), which synthetise and secrete serotonin. Therefore, we can propose that the PBN may be directly stimulated by serotonin.
As for the indirect stimulation of the PBN by serotonin, we need to delve into the model of serotonin-mediated anorexia via two distinct populations of neurons in the arcuate nucleus of the hypothalamus (ARC). In the ARC, serotonin activates Gq-coupled 5-HT2CRs on proopriomelanocortin(POMC)-expressing neurons and inhibits Gi-coupled 5-HT1BRs on agouti-related peptide(AgRP)-expressing neurons. The product of POMC, alpha-melanocyte stimulating hormone (alpha-MSH), and AgRP are endogenous ligands of melanocortin 4 receptors (MC4Rs); the former activates and the latter inhibits MC4R-expressing neurons. Therefore, upon serotonin stimulation of the two populations of neurons, the release of alpha-MSH and AgRP can be altered, leading to increased likelihood of activation of MC4R-expressing neurons. This ultimately results in the serotonin-mediated anorexia, i.e. reduction of food intake. We know that MC4Rs are essential (though not necessarily the only) for the effect of serotonin on food intake because the serotonin-induced anorexia is abolished in MC4R-knockout mice as it is for that matter in 5-HT1BR- and 5-HT2CR-knockout mice.
We also know that MC4Rs are expressed widely throughout the central nervous system (CNS) and among other sites also in the PBN (Kishi et al., 2003; Liu et al., 2003; Mountjoy et al., 1994). Furthermore, we know that the ARC projects directly to the PBN (Wu et al., 2008; Bromberger et al., 1998; Moga et al., 1990a&b; Sim et al., 1991). From that we can imply that the PBN can be also indirectly affected by serotonin via the ARC 5-HT1BR- and 5-HT2CR-expressing AgRP and POMC neurons.
Significance?
From previous research, we already know that in the brain serotonin stimulates 5-HT2CRs on POMC neurons in the ARC, which project to the paraventricular nucleus of the hypothalamus (PVH), where they stimulate MC4R-expressing neurons to ultimately reduce food intake and cause weight reduction. But the above things that I learned today have brought the PBN much more into the picture for me, expanding the complexity of the neural pathways that control appetite and body weight.
Insulin, leptin, serotonin and melanocortin pathways all meet in the ARC
Or in the longer version that insulin, leptin, serotonin and melanocortin pathways all meet in the arcuate nucleus of the hypothalamus (ARC).
Details?
Serotonin and melanocortin pathways, at least their parts, are already well established. In the ARC, serotonin acts via Gq-coupled serotonin 2C receptors (5-HT2CRs) on neurons that co-express cnorectic proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript peptide (CART). Additionally, also in the ARC, serotonin simultaneously acts via Gi-coupled inhibitive serotonin 1B receptors (5-HT1BRs) on neurons that express orexigenic agouti related peptide (AgRP). These stimulations decrease the likelihood of AgRP release at downstream melanocortin 4 receptors (MC4Rs) and increase the likelihood of release of the anorexic alpha-melanocyte stimulating hormone (alpha-MSH) at the MC4Rs. We also know that MC4Rs are critical downstream mediators of this serotonin-induced anorexia, particularly those in the paraventricular nucleus of the hypothalamus (PVH).
POMC and AgRP neurons also express leptin receptors. Leptin, similarly to serotonin, stimulates anorexic POMC neurons and inhibits orexigenic AgRP neurons to induce anorexia, i.e. reduction of food intake.
Insulin also stimulates the ARC neurons, which express insulin receptors.
Significance?
This is all important as it highlights that brain is an important regulator of how much food we eat and, secondly, because it shows the immense complexity of the neural pathway that are involved in this process.
Details?
Serotonin and melanocortin pathways, at least their parts, are already well established. In the ARC, serotonin acts via Gq-coupled serotonin 2C receptors (5-HT2CRs) on neurons that co-express cnorectic proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript peptide (CART). Additionally, also in the ARC, serotonin simultaneously acts via Gi-coupled inhibitive serotonin 1B receptors (5-HT1BRs) on neurons that express orexigenic agouti related peptide (AgRP). These stimulations decrease the likelihood of AgRP release at downstream melanocortin 4 receptors (MC4Rs) and increase the likelihood of release of the anorexic alpha-melanocyte stimulating hormone (alpha-MSH) at the MC4Rs. We also know that MC4Rs are critical downstream mediators of this serotonin-induced anorexia, particularly those in the paraventricular nucleus of the hypothalamus (PVH).
POMC and AgRP neurons also express leptin receptors. Leptin, similarly to serotonin, stimulates anorexic POMC neurons and inhibits orexigenic AgRP neurons to induce anorexia, i.e. reduction of food intake.
Insulin also stimulates the ARC neurons, which express insulin receptors.
Significance?
This is all important as it highlights that brain is an important regulator of how much food we eat and, secondly, because it shows the immense complexity of the neural pathway that are involved in this process.
One can be positively surprised when least expected
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today I learned
I did have a talk with one of the Postdocs in my lab. Initially, I only planned to explain the current experiment I was analysing but then expanded to include my general worries and recent thoughts about studying for a PhD.
It went better than expected. I had a chance to explain my attitudes, received some constructive criticism and - quite surprisingly - was told a couple of appraising sentences.
It left me pleasantly calm and I went on to analyse my data.
It went better than expected. I had a chance to explain my attitudes, received some constructive criticism and - quite surprisingly - was told a couple of appraising sentences.
It left me pleasantly calm and I went on to analyse my data.
MC4Rs are expressed solely in CNS
Or in other words that melanocortin 4 receptors (MC4Rs) are expressed exclusively in the central nervous system (CNS).
Why is this significant? Because mutations of the MC4Rs in humans are one of the most common (if not the most) single gene mutations causing obesity. And if MC4Rs are only expressed in the CNS, it supports the important role the brain has in the regulation of energy balance, hence the brain pathways are important for obesity. This is opposed to the (now mostly extinct) thinking of obesity as a disease of periphery.
Of note, other peptides expressed exclusively in the brain are POMC (proopriomelanocortin), serotonin 6 receptor, serotonin 2C receptor, agouri related peptide. All of these are also important in the regulation of energy balance.
Why is this significant? Because mutations of the MC4Rs in humans are one of the most common (if not the most) single gene mutations causing obesity. And if MC4Rs are only expressed in the CNS, it supports the important role the brain has in the regulation of energy balance, hence the brain pathways are important for obesity. This is opposed to the (now mostly extinct) thinking of obesity as a disease of periphery.
Of note, other peptides expressed exclusively in the brain are POMC (proopriomelanocortin), serotonin 6 receptor, serotonin 2C receptor, agouri related peptide. All of these are also important in the regulation of energy balance.
It is important to accept 'negative' feedback
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today I learned
During one's PhD, there will be - or should be - a lot of feedback because PhD is primarily not about getting the title but about learning how to be an independent researcher.
The feedback, which is received, shouldn't be even distinguished as positive or negative. Those words lose meaning a little. Because what is important is whether the feedback is constructive and whether it will lead to improvement of future work.
Also, I find that many of us get easily offended because we take it personally and as too critical or negative. But a more helpful approach is the above one, of objective evaluation and selecting the key message for the self-improvement.
The feedback, which is received, shouldn't be even distinguished as positive or negative. Those words lose meaning a little. Because what is important is whether the feedback is constructive and whether it will lead to improvement of future work.
Also, I find that many of us get easily offended because we take it personally and as too critical or negative. But a more helpful approach is the above one, of objective evaluation and selecting the key message for the self-improvement.
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