Lecture 8 - Chapter 23: Neuronal networks Flashcards
Name four steps that are needed to form a neuronal network.
- Establish polarisation: what part of the cell body will grow dendrites and what part will grow axons.
- Neurite outgrowth (growth and elongation)
- Synaptic contact (making contact with other cells)
- Stabilisation/disassembly of contact with other cells.
What is meant with a polarised cell?
Cell polarity refers to the intrinsic assymetry observed in cells, either in shape, structure, or organization of cellular components.
Describe why epithelium cells have polarity.
The apical side of an epithelial cell is different to its basal-lateral side. On the apical side, there’s the actin cytoskeleton and the apical endosomes. On the basal-lateral side there’s an microtubule network (that is also polarized) and the basal-lateral endosome.
What is a growth cone?
A growth cone is a large actin-supported extension of a developing or regenarating neurite seeking its synaptic target. The growth cone consists of a lamellipodium with filopedia sticking out of the lamellipodium.
How is this polarity that is visible in endothelial cells seen in neurites?
The cytoskeleton of the neurite itself is made up out of a microtubule network, while the growth cone also contains complex actin networks and filopodia.
You can compare the growth cone of a neuron to the apical side of an epithelial cell and the basal-lateral side of an epithelial cell can be compared to the cytoskeleton of the neurite itself.
How do neurites (out)grow?
By their growth cone and by reorganisation of the highly dynamic cytoskeleton. Here, microtubules from a sort of loop. This stabilizes the lamellipodium, where subsequently the filopodia extent in the direction that the neurite needs to grow to.
What neuropeptide/signal can act as an attractor for outgrowth of neurons?
BDNF
Explain how neurites (out)grow.
So there needs to be reorganisation of the cytoskeleton of the neurite. For this, there needs to be an attractive cue (like BDNF) and a repulsive cue. The attractive cue stimulates actin polymerization, while the repulsive cue stimulates actin depolymerization.
The fact that attractive and repulsive cues originate from different specific places, results in asymmetric activation of receptors. These signals (and their second messengers) determine whether its an attractive or repulsive cue.
The extracellular signals (and their second messengers) tell the cell whether its an attractive or repulsive cue. What happens when the cell determines the signal as an attractive cue? And what happens when the cell determines the signal as a repulsive cue?
- Attractive cue → exocytosis, cytoskeletal assembly and increased adhesion
- Repulsive cue → endocytosis, cytoskeleton disassembly and decreased adhesion.
Note: the components that are endocytosed, are subsequently used for exocytosis.
Fill in:
- Cyclic GMP is an important second messenger for attractive or repulsive cues.
- Cyclic AMP is an important second messenger for attractive or repulsive cues.
- Calcium signaling is repulsive or attractive or both cues.
- Actin assembly and adhesion is important for repulsive or attractive or both cues.
- Cyclic GMP is an important second messenger for repulsive cues.
- Cyclic AMP is an important second messenger for attractive cues.
- Calcium signaling is both attractive or repulsive.
- Actin assembly and adhesion is important for attractive cues.
Besides diffusable chemoattraction and chemorepulsion, there is also non-diffusable contact-mediated attraction and repulsion. What’s the importance of these contact-mediated signals?
If there’s a bundle of outgrowing axons, the contact-mediated attraction cues tell the outgrowing axons to stick together. And at the point where axons need to split up (because one axons is needed at a different place than other axons), the contact-mediated attraction switches to contact-mediated repulsion. This sends the axons away to their own specific place.
Name non-diffusable and diffusable mediators for attraction or repulsion.
- Non-diffusable → extracellular matrix adhesion molecules (integrins), ephrins, cadherins, Ca-independent adhesion molecules (CAMs).
- Diffusable → semaphorins, netrin/slits, neurotrophic factors (NFG/BDNF/neurotrophins)
What are integrins and what happens upon activation?
Integrins are transmembrane receptors that facilitate cell-cell and cell-ECM adhesion. Integrins are composed of 2 subunits (dimers) that stretch out upon activation.
Integrins can be regulated from inside and outside of the cell. What is outside regulation of integrins called and how does it work?
Outside-in signalling → growth cones encounter ECM (ligand) to which integrin can bind, which allows integrins to stretch out more. This leads to intracellular signal that enhances cell polarity, structure and gene expression.
Integrins can be regulated from inside and outside of the cell. What is inside regulation of integrins called and how does it work?
Inside-out signalling → cytoplasmic molecules like Talin can bind to the cytoplasmic tails of integrins. This stimulates cell adhesion and migration and ECM assembly.
Fill in:
- CAMs are Ca2+-dependent or -independent.
- Cadherins are Ca2+-dependent or -independent.
- CAMs are Ca2+-independent.
- Cadherins are Ca2+-dependent.
Why are CAMs and cadherins called hand-shake molecules?
There’s no receptor-ligand interation, but more or less a dimerization process. Like the fact that cadherins interact with B-catenin, which are similar to each other.
What are tyrosine kinase receptors (RTKs)?
Receptors that dimerize upon signal molecule binding. The receptor is then activated and its intracellular tail can then be auto- and cross-phosphorylated by the activated kinase domains.
What function do ephrins have?
Interaction of ephrin ligands with their tyrosine kinase receptor constitute a cell-cell recognition. Upon cell-cell recognition ephrins from one cell can interact with the ephrin receptors on the other cell and can activate a variety of cytoplasmic protein kinases.
Ephrins and Ephs activate a variety of signallig pathways and, depending on the nature of signal transduction, can be either growth-promoting or growth-limiting. When is signalling growth-promoting and when is it growth-limiting?
- Growth-promoting, when ephrin of one cell interacts with the eprhin receptor on another cell.
- Growth-limiting, when the extracellular domain of an ephrin ligand or receptor is proteolytically cleaved.
Why is there bidirectional signalling in ephrin ligand and ephrin receptor interaction?
Because ephrin interaction is dependent on the interaction of two cells. In the case that ephrin ligand of one cell interacts with ephrin receptor on another cell, in both cells intracellular cascades are initiated. Like the activation of kinases.
When ephrin ligands or receptors are proteolytically cleaved, this also influences the interaction between the two cells and thus influences the intracellular cascades.
Note: we’ve just discussed the 4 molecules (integrins, ephrins, cadherins and CAMs) that from contact-mediated attraction/repulsion → non-diffusable cues.
Now the diffusuble cues will be discussed
For what process are Netrins and Slits important for?
Crossing the midline of the brain/spinal cord. It’s seen that in a netrin null mouse, neurons do not cross the midline of the spinal cord (right picture).
Where are netrin and slits (and also semaphorins) produced and what’s the difference between netrin and slit (and semaphorins)?
Netrins and slits are produced in the floorplate of the neural tube.
- Netrin acts as a attractive signal for outgrowing neurons.
- Slit (and semaphorins) act as a repulsive signal for outgrowing neurons.
What receptors on the axon with a growth cone (i.e. the cell that is searching for signals to follow) are important for attraction or repulsive signals from netrin or slit? Also describe whether they interact with Slit or Netrin.
Robo and DCC receptor, they both project to the Rho/GAPs system that influences actin polymeres.
- DCC interacts with Netrin
- Robo interacts with Slit
What happens when Robo is knocked-down in Drosophila?
Outgrowing axons are not repelled anymore from the floorplate and it is then seen in Drosophila that neurons keep circling around the floorplate (Robo → roundabout).
Just study
What are semaphorins?
Semaphorins are another class of membrane proteins that have a role as axonal growth cone guidance molecules. Like Slit, it tells axons to stay away.
What kind of experiment was performed that researched the function of semaphorins?
They modified HEK-cells to produce semaphorins and next to it they put a dorsal root ganglium. They looked in what direction the axons of the dorsal root ganglium would grow. What you can clearly see in the picture is that axons grow on the opposite side of where the HEK cell was located.
To control if this was really the case, they also used antibodies against semaphorin, to see if the direction of the axons changed, which it did.
What is the function of Neuregulin 1? What happens weh Neuregulin 1 is active?
When Neuregulin 1 is active, the extracellular domain of the protein is cleaved and moves to the postsynaptic site to bind with ErbB receptors. ErbB receptors are tyrosine-kinase receptors and thus dimerize upon Neuregulin 1 binding. This causes downstreams signalling that stimulates the insertion of postsynaptic neurotransmitter receptors.
Does neuregulin 1 have other functions in the nervous system?
Yes! See picture.
What is important for synapse formation?
Complex machinery (some neurotransmitters that can be released and some receptors that can interact as a base for later synapse formation).
This pictures explains that the synaptic cleft is very small (few nm in diameters) and that the synatic cleft is just large enough to fit the complex machinery between.
Ok
How could you test which out of these many adhesion molecules is essential for synapse formation?
Expressing neuroligin in non-neuronal cells (HEK-cells) and then expressing different adhesion molecules. With this you can look for when synapses are being made and you can link this to the adhesion molecule that is expressed.
So we can conclude that different types of molecules are needed for synapse formation and of course if a synapse doesn’t work, it means that no signal is able to pass through. What is a way of the brain to make sure that the right collection of molecules are present for synapse formation and synaptic transmission?
A way to make sure that the right collection of molecules are present, is to have ready made active zones.
For instance, when new synapses are formed there are active zone (AZ) vesicles. When there’s synaptic connection, these vesicles fuse and deliver essential proteins that you need for neurotransmitter release (SNARE porteins, Munc-13 and Munc-18 etc.)
Name the steps in formation of pre- and post-synaptic clusters.
- First initial contact is regulated by cadherins and protocadherins.
- When a proper connection is established, there’s insertion of AZ precursor vesicles. They provide everything that is needed for neurotransmitter release.
- Neurexin makes contact with neuroligin.
- Neuregulin is cleaved and EphB is activated, which results in postsynaptic receptor insertion.
- Synapse maturation, where proper specialization occrus. Here presynaptic vesicles are assembled and postsynaptically the mushroom-shaped dendritic spine (with postsynaptic density) is formed.
What’s the difference in a developing nervous system compared to an established nervous system in adults?
- Developing nervous systems have polyneuronal innervation → multiple neuronal fibers innervating the same muscle fiber.
- During adulthood specificity is created, where one neurons innervates one fiber.
Describe an example of polyneuronal innervation and what happens after development is complete.
The axons projecting from the olive nucleus to the cerebellum are called climbing fibers and innervate the Purkinje cells in the cerebellum.
- When you’re young, there’s polyneuronal innervation, meaning that multiple nuerons innervate the same Purkinje cells. Over time, due to competition of neurons for neurotrophic factors, one neuron will win the competition from the others. The innervation then becomes more selective and extensive.
- So in adultlife, there’s only one cells that innervates one Purkinje cell in the cerebellum.
How were neurotrophins discovered?
A dorsal root ganglium was dropped into different baths with different neurotrophins. They saw that the ganglium degenerated when neurotrophin concentrations were low.
What is seen when BDNF is overexpressed in neurons?
All dendrities and axons grow out further and the neuron has a more complex phenotype.
How is it seen that neurotrophic factors modulate competition (what research was performed to confirm this)?
By putting a neuron into a bath with NGF and seperating the branches of the neuron into two different compartments (where the cell body is localized between these two compartments).
By then removing NGF in one branch/compartment and around the cell body and adding NGF to the other comparment, it can be studied how the neuron reacts.
It is seen that in the absence of NGF, neurites regress and that in the presence of NGF neurites continue proliferation of branches.
How can it be confirmed that neurotrophic factors are selective for certain neurons?
By using different neurons (dorsal root, nodose or sympathetic ganglia) and combining them with different neurotrophic factors. It can be seen that for instance the nodose ganglian does not respond to NGF, but it does respond very clearly to NT-3.
What is the reason why neurotrophic factors are selective?
By having different neurotrophins and different concentration gradients in the brain, the activity of certain types of neurons can be carefully regulated.
For example: the skin contains different nerves for pain, hair follicles, muscles etc. that each need to have their own innervating neurons. The location of these nerves for all the different components of the skin, in combination with what neurotrophins they produce, makes sure that the right axon is guided to the right nerve.
There are high and low affinity neurotrophin receptors. Name these.
- High affinity, specific receptors → TrkA, TrkB and TrkC.
- Low affinity, general → p75 receptor.
First it was thought that when a receptor is internalized, the signal initiated by neurotrophins stop. This would be because neurotrophins can’t reach their receptor anymore, once the receptors are internalized. This might not be the case.
What is seen/known now?
Sometimes, internalization is needed or enhances interactions. Because internalized receptors in endosomes are enriched with downstream signaling molecules.
