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+ BIOL 371 > Paper 4: > Flashcards

Paper 4: Flashcards

1
Q

What is appetite, energy balance, and metabolism regulated by?

A

1960s/80s: CNS neurons found in hypothalamus

1990s: peptide hormones that circulate in bloodstream

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2
Q

What neurons are found in the ARC/ARH?

A

POMC and AgRP neurons

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3
Q

What neurons are found in the LH?

A

MCH neurons (their somata)

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4
Q

What is the blood-brain barrier (BBB) formed by?

A

tight junctions between capillary endothelial cells

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5
Q

What is the blood-brain barrier (BBB) at median eminence formed by?

A

fenestrated microvessel loops, which make it leaky

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6
Q

What is the brain-cerebrospinal-fluid barrier (BCSFB) in the hypothalamus formed by?

A

tanycytes – specialized hypothalamic glia that line the floor of the third ventricle (3V)

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7
Q

Where is the brain-cerebrospinal-fluid barrier (BCSFB) leakier?

A

near ARC/ARH is leakier than elsewhere in the brain vesicles

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8
Q

What can the BBB around the median eminence be structurally altered by?

A

food deprivation, which triggers:

  • increased microvessel fenestration due to release of VEGF-A
  • enhanced permeability in both the ME and ARH
  • activation of tanycytes and B-CSF-B reorganization
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9
Q

How do we know that the signals that direct the 3 changes triggered by food deprivation (see previous question) are coming from the brain, not the blood?

A

these events also occurred if CNS glucose was reduced without changing blood glucose

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10
Q

What is melanin-concentrating hormone (MCH)?

A

neuromodulator peptide released from MCH neurons in the lateral hypothalamus (LH)

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11
Q

What can affect MCH neuron activity?

A

fasting

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12
Q

What does activation MCH neurons lead to (in relation to food)?

A

increases in food intake

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13
Q

Where are some some MCH-secreting neurons?

A

they project their axons to the ME and third ventricle

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14
Q

What can cause excessive eating or complete suppression of appetite?

A

lesions that eliminate all neurons in specific hypothalamic nuclei

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15
Q

What hormones can affect appetite?

A

peptide hormones secreted from the periphery (ie. outside the CNS) can affect appetite via actions on receptors found within the CNS

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16
Q

Where are fenestrated capillaries found?

A

(rare – NOT found throughout the brain)

in a baseline state they are found in the median eminence (ME), OVLT (osmosensing part of hypothalamus), and SON (in hypothalamus)

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17
Q

Are fenestrated capillaries critical components of the BBB?

A

no

fenestrated capillaries are holes through the capillary wall – which means there can’t be a BBB because fluid can pass through these holes

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18
Q

In a mouse that is able to freely obtain food, where is the BBB is permeable? Where isn’t it permeable?

A

permeable in the median eminence, but not in the ARC/ARH

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19
Q

What is the candidate gene approach?

A

experimental approach which creates variation in pre-defined genes of interest to assess their contribution to a specific biological mechanism

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20
Q

What is a cell-type marker gene?

A

gene expressed in a small subset of cells within a tissue, whose promoters can be used to selectively tag just that subset of cell

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21
Q

Example of how to generate variability in a candidate gene:

A

two different genetically-modified mouse lines are bred together

  • Cre: recombinase enzyme that deletes any DNA sequences it finds between LoxP sites
  • reporter gene: gene that makes readily detectable products (or confer a novel physiological property) when they are expressed
  • Rosa28: stretch of DNA in the mouse genome which is easy to insert transgenes into and get them expressed
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22
Q

What is Cre?

A

recombinase enzyme that deletes any DNA sequences it finds between LoxP sites

23
Q

What is a reporter gene?

A

gene that makes readily detectable products (or confer a novel physiological property) when they are expressed

24
Q

What is vimentin?

A

protein expressed by tanycytes

25
Q

What is lectin?

A

protein found on blood vessels, but not neurons

26
Q

Figure 1: MCH Neuron Projections in the ME

What were the observations from this figure?
What was the conclusion?

A

observations:

  • MCH-releasing neurons have neurites near tanycytes
  • MCH-releasing neurons axon terminals are very near vimentin processes and capillary loops (lectin)

conclusion:
- MCH neurons likely make synapses on tanycytes and ME capillaries

27
Q

What is chemogenetics?

A

introduction of artificially engineered signalling molecules/receptor systems into cells of interest to enable targeted control of their activity

28
Q

What is a common type of chemogenetics?

A

introduction of DREADDs such as hM3Dq

29
Q

What is hM3Dq?

A

mutated form of the M3 muscarinic receptor (Gαq-coupled) which is incapable of binding to AC

can instead be activated by clozapine-N-oxide (CNO) – artificial ligand which does not bind to any native (non-mutated) GPCRs in a mouse brain

30
Q

Will hM3Dq act as an inhibitory or an excitatory DREADD?

A

neurons become more active (depolarizes) via CNO

31
Q

Figure 2: Chemogenetic Activation of MCH Neurons Enhances Permeability of ME Barrier

What did this figure show?

A

chemogenetic activation of MCH neurons increases permeability of ME barrier
- more dye can be seen (dye was originally only in bloodstream

32
Q

Figure 3: Chemogenetic Activation of MCH Neurons Enhances Permeability of ME Barrier Through Increased Fenestration of Micro-vessel Loops

What was the observation of this data?
What was the conclusion?

A

observation: number of MECA-32-containing microstructures within ME and Arc is increased in hM3Dq^MCH mice
conclusion: chemogenetic activation of MCH neurons causes increased capillary fenestration at the B(H)B but does not alter the tanycytic B-CSF-B

33
Q

Figure 3: Chemogenetic Activation of MCH Neurons Enhances Permeability of ME Barrier Through Increased Fenestration of Micro-vessel Loops

What was the observation of this data?
What was the conclusion?

A

observation: number of MECA-32-containing microstructures within ME and Arc is increased in hM3Dq^MCH mice
conclusion: chemogenetic activation of MCH neurons causes increased capillary fenestration at the B(H)B but does not alter the tanycytic B-CSF-B

34
Q

Stimulation of MCH neurons promotes the action of what hormone?

A

leptin

35
Q

What is leptin?

A

hormone that has some ability to suppress appetite – at least in naive mice (mice that lack leptin)

36
Q

Figure 4A-B: Chemogenetic Stimulation of MCH Neurons Promotes Leptin Action

Why do you think the authors are using leptin for these experiments on food intake, rather than another peptide hormone (ie. ghrelin)?

A

because leptin is usually not present in fasted mice and thus its effects are not complicated by the need to account for endogenous levels of the hormone
- leptin is being used because it shouldn’t be present, so they can control how much leptin is in the bloodstream (which will only be present if the researchers put it in)

because leptin is capable of suppressing appetite, which is easy to measure in a fasted (and thus hungry) mouse

  • mouse that has been fasted is motivated to eat and not do much else
  • if you try to test something else, mouse might have trouble performing because it is just looking for food
37
Q

Figure 4A-B: Chemogenetic Stimulation of MCH Neurons Promotes Leptin Action

What were the observations of this figure?
What was the conclusion?

A

observations:

  • WT mice do not eat less when injected with leptin vs. when injected with saline after 16 hr of fasting
  • fasting MCH-hM3Dq mice show reduced appetite (food intake) after leptin treatment

conclusion:
- activating MCH neurons leads to increases in the animal’s sensitivity to a peptide hormone (leptin) known to have receptors in the ARH

38
Q

What is optogenetics?

A

introduction of light-activated ion channels into neurons to enable targeted control of their activity (by illumination using laser light of specific wavelengths)

39
Q

What is channelrhodopsin (ChR2)?

A

non-selective cation channel derived from algae that is the most common optogenetic tool for stimulating neural activity

small fibre-optic cables implanted inside CNS allow for optrodes (light equivalent of electrodes) to stimulate axons near the optrode which express ChR2

40
Q

Are there inhibitory forms of both chemogenetic DREADDs and optogenetic ion channels that can silence neuronal AP firing when activated?

A

yes

41
Q

Explain one advantage of using optogenetics instead of chemogenetics to study processes going on in the brain.

A

no systemic adverse effects

pharmacology is almost always messy, so if there are any negative side effects of CNO, optogenetics will minimize that

42
Q

Explain one advantage of using chemogenetics instead of optogenetics to study the same processes.

A

easier to set-up, and requires less precision – to do chemogenetics, you just need to give the mouse one injection at the right time, or you can put it in their water bottle (non-invasive)

to do optogenetics, you need to get an optrode into the brain, and this requires complex brain surgery

  • need to make sure it’s placed in the correct part of the brain and that you don’t miss
  • make sure you don’t cause seizures or brain bleeds
  • head has wire attached to it sticking out, which may make it difficult for mice to perform certain tasks and behaviours
43
Q

Figure 5A-B: Optogenetic Stimulation of MCH-ME Projections Promotes Leptin Action

What did this figure show?

A

(like with hM3Dq) activating MCH neuron terminals at ME leads to more diffusion of dye into the ARH

44
Q

Figure 5C-E: Optogenetic Stimulation of MCH-ME Projections Promotes Leptin Action

What did these figures show?

A
  • optogenetic activation of MCH neurons is associated with an increase in fenestrated capillaries (observation)
  • optogenetic activation of MCH neurons causes an increase in leptin sensitivity
45
Q

Supplementary Figure S6A-B: (Related to Figure 6 and 7) MCH Does Not Alter Tanycyte Signaling

What do these figures show?

A

shows why the authors no longer discuss tancytes in the main paper

  • tanycytes do not respond to MCH because they do not have MCH receptors
46
Q

What is the unbiased screen approach?

A

experimental approach where the expression/activity of all genes (or as many as possible) is compared simultaneously to look for molecules that could be associated with the biological process being studied

  • do not make any assumptions (any formed hypotheses)
47
Q

What does single cell RNA-seq (scRNA-Seq) allow?

A

allows you to classify cells by a large set of different genes they express, instead of by just one individual peptide they might secrete

  • mRNA is extracted from each cell, genetically ‘bar-coded’ and copied into stable double-stranded complementary DNA (ds-cDNA)
  • ds-cDNA is sequenced using high-throughput sequencing – number of counts for each gene sequence are determined for each cell
  • pattern of gene expression levels in different cells is used to classify cells and to identify all the genes expressed in known cell types
48
Q

Figure 6A-B: Single-Nucleus Sequencing of MCH Neurons

What was the observation of this figure?
What was the conclusion?

A

observation: cluster 0 (one fo the 4 major neuron subtypes) expresses a large number of genes that are involved in regulation of VEGF-A signalling pathway
conclusion: data suggests that some MCH neurons could be releasing VEGF-A directly (rather than via stimulating the tanycytes)

49
Q

Figure 7A-C: VEGFA Expression in MCH Neurons

Why do the authors use immunohistochemistry as well as mRNA expression?

A

mRNA is not always transcribe into protein – only proteins can have signalling function

50
Q

Figure 7A-C: VEGFA Expression in MCH Neurons

Why do the authors use in situ hybridization?

A

to confirm that it is the MCH-expressing cells in the lateral hypothalamus (LH) expressing mRNA for VEGF-A

51
Q

Figure 7A-C: VEGFA Expression in MCH Neurons

Why do the authors use immunochemistry?

A

to show that VEGF-A protein is present in MCH neurons

52
Q

Figure 7D: VEGFA Expression in MCH Neurons

What is Axitinib?

A

specific blocker of VEGF receptors

53
Q

Figure 7D: VEGFA Expression in MCH Neurons

What was the observation of this figure?
What was the conclusion?

A

observation: fasting mice with activated MCH neurons did NOT show significant appetite suppression by leptin if they had also been injected with Axitinib
conclusion: MCH neurons act through VEGF-A to induce capillary fenestration and open B(H)B to circulating peptides

54
Q

Summary

A

this paper combines many many modern techniques to test a hypothesized candidate mechanism for increases in B(H)B permeability during fasting (that hypothalamic MCH neurons activate tanycytes, causing capillary fenestration and tight junction reorganization by the release of tanycytic VEGF-A)

when the hypothesized mechanism is not supported by the experimental data, they switch to an unbiased screen approach using single cell RNA sequencing to generate data leading to a revised hypothesis (that a subset of MCH neurons release VEGF-A from axon terminals when activated, which directly causes the observed increase in capillary fenestration)

+ BIOL 371 (22 decks)

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