Flashcards in BMS381 Developmental Neurobiology Deck (347)
What is the purpose of the central nervous system?
The Central Nervous System allows us to receive information from our environment both externally (from the peripheral nervous system) and internally (e.g. release of stress hormone)
How did nervous systems first arise?
- Nervous systems arose with multicellularity, allowing a variety of neurons to give flexibility and coordination
- Early neural cells arose from the surface layers – ectoderm differentiates into skin or neural tissue
What is the neuroepithelium?
Neuroepithelium is a one cell thick sheet of neural tissue. It is already fated to be neural meaning it can give rise to a neurone or a glial cell
What is the neural tube?
A single layered neuroepithelium, induced by the ectoderm on the dorsal side of the embryo
This then grows and elongates along the AP axis and rolls into the neural tube
Simple cell movements that underlie these morphological changes
What factors can govern gene expression?
Gene expression in a cell can be governed by extrinsic factors (morphogens) and intrinsic factors (transcription factors)
How does ectoderm acquire a neural fate?
Under the influence of transcription factors such as Gsc, the organiser expresses unique secreted products that are all antagonists of the BMP signal (chordin, noggin)
This causes the phosphorylation of SMAD157 and therefore the up regulation of transcription factors such as SoxD
This induces a neural fate in those cells
How does ectoderm acquire an epidermal fate?
BMP is not inhibited meaning SMAD157 is not phosphorylated
Indues transcription factors such as Msx1 and GATA1
Causes epidermal differentiation
What occurs in gastrulation?
As soon as the organiser induces the neural plate, the organiser self-differentiates into the axial mesoderm. The axial mesoderm then involutes and undergoes convergent extension
How does gastrulation lead to the formation of the AP axis?
- The first cells to migrate are the anterior endoderm cells, followed by the prechordal mesoderm cells. Thy migrate up and along These mark the future anterior of the embryo and will be important in building the head of the embryo
- The last cells to migrate are the notochord. These will underlie most of the body with the back end forming the posterior end of the embryo
How does gastrulation lead to the formation of the DV axis?
The axial mesoderm now underlies prospective neural tissue which is a dorsal structure. This is why the organiser is initially referred to as marking the future dorsal axis as well as the anterior/posterior axis
What does impression mean?
Individual cells leave an epithelial sheet and become freely migrating mesenchymal cells
What is epiboly?
A sheet of cells spreads by thinning
What is intercalation?
Rows of cells move between one another, creating an array of cells that is longer but thinne
How do we know that the early induced neural plate is anterior in character?
The organiser self-differentiates and undergoes convergent extension. If we experimentally stop development at this point in time, and look with molecular markers, we find that the neural plate is expressing markers that are later confined to the forebrain (telencephalon and diencephalon)
How does the neural tube develop its anterior posterior identity?-
- BMP antagonists and Wnt antagonists are maintained anteriorly in the prechordal mesoderm
- FGF, Wnts, RA are expressed posteriorly by the late organiser/ Node, promote growth and posteriorised
- Formed by establishing a regional pattern by placing two antagonistic molecules at each end of a forming (growing) structure
What does the neural crest give rise too?
The entire PNS
How are neural crest cells formed?
- BMPs are expressed by surface ectoderm cells, next to edges of induced neural plate
- Neural plate border cells are established by intermediate levels of BMP signalling triggering transcription factors (msx). These either develop into neural crest cells or are retained at the border to form roof plate cells
- BMPs, working with Wnt and Fgf signaling, initiate a cascade of events that will give rise to highly-potent, proliferative Neural crest cells
How does dorsalisation of the neural tube occur?
- Neural crest cells and roof plate cells are induced by the neural plate boarder cells
- Roof plate cells then upregulate BMPs
- Secreted BMPS diffuse into the dorsal neural tube. They induce expression of a transcription factors (Pax6, Pax7, Pax3, Lim1) that cause cells to acquire ‘dorsal identities’ and form dorsal progenitors
How does ventralisation of the neural tube occur?
- The axial mesoderm (notochord) start to produce Shh which acts as a morphogen
- This induces the floor plate which then also starts to secrete Shh – positive feedback.
- The Shh diffuses out of the floor plate and notochord and into the neural tube
- Induces the transcription factor Gli1 turning on expression of other floor plate genes – e.g. Shh itself
- Genes encoding different transcription factors begin to be transcribed and translated in different cells along the D-V axis
How can the role of Shh as a morphogen be investigated?
What are homeodomain transcription factors?
Master regulatory transcription factors
What was the purpose of the paper 'Identification of a neural crest stem cell niche by spatial genomic analysis ' in 2017?
- The neural crest is an embryonic population of multipotent stem cells that form numerous defining features of vertebrates
- Due to lack of reliable techniques to perform transcriptional profiling in intact tissues, it remains controversial whether the neural crest is a heterogeneous or homogeneous (single stem cell) population
- This paper describes a novel technique that combines in situ hybridization with machine learning to examine complex gene expression in cells in the developing neural tube at single cell resolution
How did the paper 'Identification of a neural crest stem cell niche by spatial genomic analysis' in 2017 allow advancements in the field?
They were able to examine the expression of 35 genes at a time. Before this could only happen with two genes. This is much cheaper and efficient. Advances in microscopy allowed better resolution and the ability to visualise individual transcripts as a single dot
What is hierarchically clustering?
When a computer is fed large data sets and the computer sorts the data and categorises into the group
What did the paper 'Identification of a neural crest stem cell niche by spatial genomic analysis ' find?
Used hierarchically clustering.
- The clustering revealed five distinct groups within the chicks dorsal neural tube.
- They pooled data together from three embryos and discovered two main cell populations
- Cells that express both pluripotency and differentiation markers and cells without a pluripotent signature
- These can be further clustered into different subpopulations of neural or neural crest cells. They express different stem cell markers
How is the majority of the CNS structured?
In columns and layers
Why is the majority of the CNS structured in layers ad columns?
- The development of the AP and DV axis
- Spatial morphogens on opposite sides of the spinal cord (eg. BMP, Shh) set up opposing concentration gradients causing differentiating cells to move laterally and form layers
- Time - Neurogenesis occurs in waves forming different layers per wave
What part of the CNS does not conform the column and layer structure?
How are the neurones of the hypothalamus structured?
Neurones are found in clusters called nuclei that are arranged in a patchwork manner
What is the function of layers in the CNS?
Allow organisation of function
Is there any organisation of function in the hypothalamus?
In each nucleus, there are clusters of neurones which can have completely opposite functions
What are the functions of the hypothalamus?
Controls the core body functions
- temperature, growth, electrolyte balance, metabolism, reproduction, sleep, stress (cortisol)
Maintains mental and behavioural homeostasis
- Desires, Mood, Anxiety, Sexual drives, Motivation, Trust, Aggression, Stress
What was first discovered about the development of the hypothalamus?
Shh loss of function studies (1996)
Shh knockout lead to dismorphology of the forebrain, cyclopia and holoprosencephaly
Lead to the understanding that Shh was important in the development of the ventral part of the brain including the hypothalamus
What part of the axial mesoderm is thought to be involved in hypothalamus development?
This focussed attention on the prechordal mesoderm, since this part of the axial mesoderm expresses Shh and underlies the anterior most part of the neural tube
The prechordal mesoderm doesn’t extend all the way to the front of the neural tube
A number of experiments showed that Shh deriving from the prechordal mesoderm induces a fan shaped set of cells in the neural plate immediately above it that themselves express Shh. What experiments could be used to show this?
Surgically ablate the pm: expect to see no shh induced
Graft extra pm: expect to see ectopic shh
Combine a piece of pm with a piece of naïve neural plate: shh induced
Same as 3 but pre-treat pm to block shh: should prevent effect
Conditional knock-down of shh in pm in vivo
What did the finding that the prechordal mesoderm induces a fan liked group of cells that express Shh focus attention on and why?
A part of the ventral midline of the forebrain on a ‘floor plate’ like structure
- The fan shaped Shh expressing cells stop abruptly at the end of the floor plate like structure (yellow)
How did the discovery of the fan like group of Shh secreting cells provide a theory of the unusual arrangement of nuclei in the hypothalamus?
- Shh will diffuse from the Shh producing cells. The midline cells secreting Shh will pattern across the AP axis (normal layer and column organisation) but the fan shaped set of cells at the end of the Shh secreting cells will cause Shh to diffuse in a circular pattern around the fan, patterned the DV axis as well as the AP
- Could the pattern of hypothalamic development be explained by the formation of arcs of progenitor territories, around the end of this floor plate.
Was the theory of - the fan like cells secreting Shh in an arc pattern being responsible for the interesting hypothalamic arrangement - correct?
- For a very brief period (0.5 days in chick), you do see these patterns. But they become very rapidly obscured
- Analysis of known progenitor markers at the end of the floor plate showed chaotic clustering around the edge of the ‘floor plate-basal plate’ territory.
- These patterns could not be explained by a simple Shh morphogen gradient or arcs of progenitors
How does the gene expression in the prechordal mesoderm change during embryogenesis?
1. Expresses Shh - induces the set of fan-shaped ‘neural-plate’ like cells above it
2. Expresses BMP antagonist (chordin, noggin, follistatin) whilst it expresses Shh to maintain anterior identity.
3. It then loses the BMP antagonists and then upregulates BMPs.
What does the expression go BMPs in the prechordal mesoderm lead to?
- BMPs diffuse into the fan-shaped ‘neural-plate’ like cells and induce transcription factors called Tbx2, BMP2 and BMP7, Wnts, and Fgf10.
- They also upregulate all of the components which allow response to these signalling factors.
- Very rare to see this combination of factors (other place is neural crest cells)- induces a population of cells which will have stem cell properties.
What was the hypothesis for the experiment completed by Fu et al in 2017?
The prechordal mesoderm induces a hypothalamic stem cell that co expresses Shh, BMP4 and Fgf10
How did Fu et al (2017) test their hypothesis?
- They tested this by performing fate mapping studies to ask if cells secreting Shh, FGF10 and BMP7 give rise to many other hypothalamic cells and whether they self-renew (are they stem cells?)
- If you inject floor plate cells with a dye, it stays there – indicates that they don’t give rise to new cells/migrate and proliferate.
- If you inject same amount of dye into fan-shaped neural plate like cells, then you see it diffuse quickly into many areas of the hypothalamus-gives rise to many cell types and so is multipotent
What did Fu et al (2017)s experiment find?
Found that the hypothalamus derives from the Shh, BMP7 and FgF10 secreting cells that lie above the prechordal mesoderm
- As if you inject dye into fan-shaped neural plate like cells, then you see that these cells give rise to a large amount of the hypothalamus
Why did Fu et al (2017) think that the Shh, BMP7 and FgF10 secreting cells that lie above the prechordal mesoderm are stem cells?
Because this combination of signals is similar to neural crest (although NC doesn’t have Shh)
Why was the first experiment that Fu et al (2017) completed not sufficient to prove the cells were stem cells?
The experiment shows that the cells can differentiate and give rise to multiple cell types but it doesn't show that the cells are self renewing
How did Fu et al (2017) show that the Shh, BMP7 and FgF10 secreting cells were self renewing?
- They injected red and green dye into the progenitor cells.
- There was always an area that appeared yellow as some of the descendants of the green cells and red cells remained in the domain – this domain continues to express Shh and FGF10
- This suggests that they are self-renewing and therefore a stem cell
What did fate mapping and marker analysis show about how stem cell differentiate and self renew?
- Cells grow out of the progenitor domain in one direction and other cells grow out in the opposite direction. While this happens, the central population is maintained as stem cells
What is meant by there being a proliferation front at the edge of each cell type domain?
There seems to be a proliferation front at the edge of each domain- therefore the highest amounts of proliferation occurs in cells that are downregulating Fgf10 and upregulating Shh
How is thought that stem cells differentiate into two different daughter cells?
- Stem like cell divides to give two daughters which look completely different – one retains Shh and other Fgf10 and BMP
- Shh is then secreted from that daughter cell to act on the other daughter cell to reregulate Shh
- This causes the daughter cell to become a stem cell again (self renews).
What is believed about the daughter cell of a stem cell that expresses FGF10 and BMP before becoming stem like again?
It is believed that the daughter cell that expresses FGF10 and BMP is a stem cell in a state of flux while it differentiates.
This is before the other daughter cells reinduces Shh so that it becomes identical to the mother cell (stem cell).
Why is there a population of Fgf10+Shh+ cells maintained throughout the life of the hypothalamus?
There are waves of neurons born in the hypothalamus throughout life from these progenitors
Explain how the anterior pituitary gland is formed
- The prechordal mesoderm induces the FgF10+Shh+ cell population and it moves away.
- In its place, there is an ingrowth of oral ectoderm which develops in and under the FgF10+ expressing cells, developing a pouch like structure.
- This Fgf10 and Shh signals to the ectoderm pouch cells telling them to upregulate a transcription factor called Lhx3.
- An ectoderm cell that expresses Lhx3 becomes Rathke’s pouch
- This will self-differentiate into the anterior pituitary gland
Explain how the posterior pituitary gland is formed
Some of the other FGF10+ cells give rise to a ventral out pocketing called the infundibulum. The distal cells of this out pocketing will self-differentiates into the posterior pituitary (diagram D) and the proximal cells will give rise to the median eminence
How is the median eminence formed?
The proximal cells of the infundibulum give rise to the median eminence
Give evidence for the involvement of Shh in the formation of the pituitary glands
Fu et al, 2017
- Wild type mouse and one treated with cyclopamine (disrupt Shh signalling)
- One treated with cyclopamine, Lhx3 isn’t there so Rathke’s pouch doesn’t form, infundibulum doesn’t form
- Results in a non-viable embryo
Why are the two pituitary glands often talked about together?
Both pituitary glands share the ability to release hormones but the hormones are released in a very different way
What is different about the way the anterior and posterior pituitary glands release hormones?
The posterior releases hormones made in neurosecretory neurones directly into the blood stream but the anterior secretes the hormones in response to a hormone releasing hormone from the hypothalamus
Give examples of hormones secreted from the posterior pituitary gland
Vasopressin and oxytocin (trust hormone)
How do axons from the hypothalamus reach target cells?
FgF10 guides them
How are hormones released from the posterior pituitary gland?
Neurosecretory neurones located in paraventricular nucleus produce vasopressin and oxytocin directly and are then transported to the posterior pituitary. Vasopressin and oxytocin (trust hormone) are then released into the blood supply of the posterior pituitary.
How are hormones released from the anterior pituitary gland?
- Another set of hypothalamic neurons project to median eminence
- They are neurosecretory and release hormone releasing neurohormones which are located in the arcuate nucleus and project to hypophyseal portal system in anterior pituitary
- These hormones are released into the portal capillary supply and travel to the anterior pituitary gland where they govern the release of hormones from endocrine cells
How do the difference cell types in the anterior pituitary control which hormone is released?
There are six different cell types make up the anterior pituitary. Each cell type is under the control of a specific neurone in the hypothalamus. E.g. Growth hormone is released from anterior pituitary when it detected growth hormone releasing hormone from the hypothalamus
What are tanycytes?
Radial glia in the hypothalamus
How are tanycytes formed?
At the proximal part of the infundibulum there are FGF10+ cells maintained as radial glia called tanycytes into adulthood
What is the role of tanycytes?
Some of these line the median eminence and are involved in acute homeostasis and others are FgF responsive multipotent and neurogenic stem and progenitor cells.
Give an example of how tanycytes can be used in acute homeostasis
- The release of growth hormone is controlled by growth hormone releasing hormone which is made by hypothalamic neurones throughout the day.
- These neurones project to the median eminence where the tanycytes block its release into the portal capillary.
- At night, the tanycytes retract and growth hormone releasing hormone can enter the anterior pituitary and growth hormone can be released. Therefore, only grow at night.
What first made us expect there was a stem cell in the hypothalamus?
The hypothalamus must be able to adapt to anticipate and meet new changing conditions (e.g. puberty, growth, pregnancy, hibernation)
- Key points in life need to generate new neurones to meet its needs
How was the distribution of tanycytes investigated?
Mouse model organsim
- dissect brain
How are tanycytes arranged in the hypothalamus?
- The hypothalamus is arranged around the third ventricle of the brain and tanycytes extend around the third ventricle forming the region
- They have long basal extensions.
- Some tanycytes project into the median eminence itself and others project to particular nuclei in the hypothalamus
What is the arcuate nucleus?
Nucleus in the hypothalamus which contain neurones that control energy balance and reproduction
How do tanycytes monitor the bodies internal environment?
- The tanycytes that project to the median eminence are in physical contact with fenestrated capillaries meaning it can sample the blood and look at the internal state
- Also sits next to the ventricle which is full of cerebrospinal fluid which is also full of signals about the internal body conditions
- The hypothalamus can then act accordingly
- The hormones that have been released into the blood can then act negatively back on the tanycytes to stop its release
Why is thought that tanycytes could be stem cells?
Tanycytes look like radial glial cells, look like they could be in a niche with blood capillaries (stem cell niches are heavily vascularised)
How can we test if tanycytes are stem cells?
Test of new cells are born in the adult
- Add a marker for S phase using bromodioxyuradine (BRDU). This acts as an analogue for thymine in DNA replication. This new DNA is then labelled so can see if cell division has occurred and therefor new cells are born
- Inject BRDU into third ventricle of a mouse and see if there are any new neurones that have formed when dissect its brain
- Found that there was de novo neurogenesis in the adult hypothalamus
- This happened in response to acute physiological changes and stressed conditions e.g. high fat diet
Does the fact that because tanycytes are dividing and new neurones are forming prove that tanycytes are stem cells?
- Doesn’t prove that tanycytes are the new neurons origin
How can you prove that new neurones forming in the hypothalamus are from tanycytes?
Genetic lineage tracing (cre loxp)
How do you make a conditional knockout?
- Genetically engineer the gene you want knocked out so that it has two flox (flanked by lox) sequences on either side of it.
- Introduce this DNA construct into the embryonic stem cells of the mice. The mouse is now carrying this gene in every cell in its body
- Take a second gene that is only expressed in the tissue interested in and clone the cre recombinase enzyme gene downstream to the genes promotor to make a second transgenic mouse
- Bread the two-transgenic mouse together
- The offspring will then remove the gene in cells that express the tissue specific gene as cre recombinase will be activated and cleave at the lox sites
How can cre lox be sued for tissue specific lineage tracing?
- Produce a transgenic mouse that has a gene expressed in tissue of interest upstream to cre
- Make a second mouse with a reporter gene downstream of a constitutive promotor but upstream to the reporter gene (makes the cell blue) add a stop codon to stop transcription of the reporter gene
- Cross the two mice. The stop sequence is recombined out where the tissue specific gene is expressed meaning the cell will appear blue
- These cells descendants will therefore all be blue
Why is tissue specific lineage tracing more effective then injecting a dye?
Because the colour will not be diluted out after each division
What is meant by temporal conditional techniques?
Able to decide then the gene is knocked out as well as in which tissues
How are temporal conditional techniques carried out?
- Cre recombinase is fused to a mutant oestrogen ligand binding domain (ERT2) that requires the presence of tamoxifen for activity
- This means that Cre is only activated when tamoxifen injected into mice
How did Robins et al, 2013 use tissue specific lineage tracing in tanycytes?
- There are different subsets of tanycytes. Specifically interested in ones that express FGF10. These are alpha tanycytes
- GLAST is a gene expressed in this subset of tanycytes
- Fuse CreERT2 downstream to the promotor of GLAST and cross with a transgenic mouse that has a Flox stop codon upstream to a reporter gene. The reporter gene could be lacz or GFP
- When tamoxifen is administered, reporter is expressed in tanycytes
What did Robins et al, 2013 discover from tissue specific lineage tracing of tanycytes?
That tanycytes are neurogenic
- The alpha tanycytes that were investigated can self-renew and give rise to other tanycytes subsets, to neurones and to astrocytes
- Will usually only produce new neurones in response to change in environment
Why doesn't tissue specific lineage tracing of tanycytes prove that they are stem cells?
Can’t conclude that tanycytes are stem cells because we can’t say that the new cells were formed from one specific tanycyte as the lineage tracing was carried out on a population of tanycytes
What did Robins et al, 2013 do to prove that one tanycyte can give rise to a population of different cell types?
Complementary in vitro studies
- Hypothalamus was dissected out and dissociated into individual cells
- Plate each cell to one well
- If it is a stem cell it must be able to proliferate. After incubation, the cell differentiated to form a neurosphere.
- The neurospheres were tested for stem cell factors.
- Also added other factors to see if it gives rise to different cell types
- Found that there are single cells in the hypothalamus that can self-renew and give rise to different cell types in vitro
What hypothalamic nucleus was found to differentiate form tanycytes?
Why is the fact that the arcuate nucleus differentiate from tanycytes give evidence to support them being stem cells?
The arcuate nucleus is responsible for controlling food intake (NPY), growth (GHRH) and reproduction (DA)
All these processes need to be able to adapt to changes in the environment
Why is thought that tanycytes derive from the FGF10+ embryonic multipotent hypothalamic progenitor?
Because they express FGF and they proliferate in response to elevated FGF
What is the idea regarding formation of new neurones involved in body homeostasis?
Emerging idea is that new neurones involved in energy homeostasis can be generated in adulthood from an Fgf-responsive/Fgf10-expressing population. This is important in changing life course
What mechanisms have been investigated in the spinal cord that are also relevant in the hypothalamus?
- What happens between the producing-responding cell: Control of Shh spread
- What happens in the nucleus: how transcription of Shh is governed
Explain the Shh mechanism
- Shh binds to the receptor patched and interacts with co receptors: Hhip, Gas1
- Patched represses the activity of smoothened
- Shh is transcribed and released from the cell. It binds to patched leading to the alleviation of the negative repression of transcription in the nucleus
- Smoothened activates the Gli transcription factors that turn on the expression of several genes
- Turns on the transcription of patched itself and other co receptors and downregulates BOC and GAS1. This leads to huge change in the cells ability to respond. This is called ligand dependant antagonism.
What is ligand dependant antagonism?
When a molecule turns on the expression of receptors that are required to respond to it. This leads to huge change in the cells ability to respond
How does ligand dependant antagonism allow for a steep diffusion gradient over a short distance?
- Usually, there would be more Shh molecules then there are patched receptors meaning that Shh can diffuse to other cells and activate transcription in those cells.
- However, Shh activates transcription of more patched receptors. (ligand dependant antagonism)
- The more molecules of patched on neighbouring cells, the less likely it is that Shh will diffuse to far away cells meaning they won’t be activated.
- This leads to non-cell-autonomous Shh pathway inhibition in cells distal to the Shh source.
How would happen if ligand dependant antagonism stops?
If a set of cells stops expressing Shh and Shh receptors then ligand dependent antagonism is stopped in that region. This will support the continual spread of Shh into neighbouring territories. Because Shh induces its own expression, this will support a spread of Shh expression to the neighbouring territory
How does ligand dependant antagonism play a role in Shh patterning of the hypothalamus at the mRNA level?
- BMP antagonists induce genes such as Pou and Sox which are transcription factors that bind to enhancers and turn on neural genes. These genes (pax7) will then be expressed throughout the ventricular zone. This activates many downstream transcription factors
- Shh expressed from notochord and floor plate which activates homeodomain transcription factor. This GliA- activated homeodomain TF binds to the enhancer of the neural genes and acts as a repressor, stopping the transcription of pax7.
What is SBE2?
An enhancer element for Shh in the hypothalamus
How was SBE2 discovered?
Doug Epstein found that transcription of Shh gne is regulated by a different enhancer element in the hypothalamus then it is in the future spinal cord/hindbrain and midbrain
- Found enhancer element called SBE2. If mutated this element then it lead to regulatory issues of Shh in the hypothalamus
What is the role of Tbx2 in the expression of Shh in the hypothalamus?
Normally, GliA and Sox2 is required for Shh expression
- However, when Tbx2 protein is present in cells, it binds to the SBE2 enhancer causing the displacement of Sox2. This means that transcription of Shh ceases.
Explain the changes expression of Shh that occur during the development of the hypothalamus?
- Shh from early prechordal mesoderm induces GliA and therefore induces Shh transcription
- Later in development, BMP7 is expressed from the prechordal mesoderm and induces Tbx2. This therefore downregulates Shh
How can the discovery of how Shh is regulated in the hypothalamus explain how the key hypothalamic progenitor cells are maintained into the adult life?
The current working model
- Shh induces a Shh+ cell at the ventral midline.
- The prechordal mesoderm properties change and begins to upregulate BMP7 which upregulates Tbx2.
- At this point in time, ligand dependant antagonism is still occurring meaning Shh can’t diffuse very far.
- Tbx2 downregulates Shh and in turn downregulates patched, preventing ligand dependant antagonism.
- This allows Shh to diffuse to the next cell along.
- The neighbouring cell is the FGF10+ progenitor cell. Shh can then be induce into that cell reforming the parent progenitor cell
Give two papers that show the importance of early developmental steps in adult formation?
Liu et al (2017)
Liu, et al (2015)
Give a brief overview of Liu, et al (2017)
- There are a number of wake promoting neurones characterised when this paper was published. However, there wasn’t much information on sleep promoting neurones
- A subset of GABA expressing neurones in the ventral zona incerta of the hypothalamus express another homeodomain transcription factor which is activated by sleep pressure
- They project to the hypocretin neurones and inhibit them
How did Liu, et al (2017) show that these Lhx6-positive GABA releasing neurones were sleep promoting?
They conditionally deleted Lhx9 in the adult mouse. Saw that the mice do not sleep
Give a brief overview of Liu et al (2015)?
- Narcolepsy is a disease that causes the patient to fall asleep often
- Patients with this have been found to have very low levels of a neuropeptide called hypocretin
- Test if Lhx9 is involved in hypocretin induction by gain and loss of function experiments
- This paper gives therapeutic applications for treatments for narcolepsy
What methods did Liu et al (2015) use to isolate hypocretin expressing cells?
Make a transgenic zebrafish in which the hypocretin promotor is linked to red fluorescent protein
Isolate cells using FACS machine which separates fluorescent cells from non fluorescent
How did Liu et al (2015) test if Lhx9 is sufficient for hypocretin specification?
Cloned each gene downstream of a heat shock inducible promotor, Hypocretin positive cells appear when the embryos are heat shocked (induce Lhx9 in all cells).
How did Liu et al (2015) test if Lhx9 is necessary for hypocretin specification?
Knockdown of Lhx9 using Cas9
How did Liu et al (2015) test if Lhx9 directly promotes hypocretin expression?
- Double fluorescence in situ hybridisation against Lhx9 and hypocretin on zebrafish embryos one hour after heat shock. Observed Lhx9-overexpressing cells
- Found in another system that Lhx9 turns on the transcription of another gene. In that paper, they worked out the enhancer that Lhx9 binds to. They therefore looked for similar DNA sequences upstream to hypocretin and mutated it to see if it is unable to activate transcription of hypocretin gene
How do the papers Liu et al (2017) and Liu et al (2015) explain how the hypothalamus works?
The hypothalamus consists of paired neurones that have opposing functions and our behaviour is regulated by the balance of these neurones. These papers are examples of this as hypocretin is the neurone responsible for wakefulness
Why do Liu et al (2017) and Liu et al (2015) show the importance of early development in adult function?
The sleep promoting and inhibiting neurones are induced by Shh
What is Lhx1 responsible for?
Responsible for suprachiasmatic nucleus which is the master regulator of the circadian cycle
What are axons?
Axons are long and carry information away from cell body in action potentials. Their termini contain neurotransmitter loaded vesicles and form synapses with the dendrites of other neurons
What are dendrites?
Dendrites are shorter and usually form a dendritic tree allowing the integration of incoming information from multiple other neurones
What is meant by neurones having polarity?
Polarity in this case refers to neurones having different structures at each end
How do the microtubules of axons and dendrites differ?
- Mature axons have highly polarised microtubules all of which are orientated in the same direction with their plus ends facing the axon terminal
- Dendrites also have microtubules but these are less ordered and have mixed orientations
What are microtubule-associated proteins (MAPs)?
Accessory proteins that stabilise microtubules by cross linking the separate strands and inhibiting polymerization
What microtubule-associated proteins are used by axons and dendrites?
Axons - Tau
Dendrites - MAP2
How can axons and dendrites be distinguished between?
The different MAPs can be used as molecular markers to distinguish between axons and dendrites
Give examples of proteins that show the compartmentalisation of the plasma membrane in neurons?
- L1 is a cell surface adhesion molecule that is restricted to axons, whereas the glutamate receptor component, GluR1, is restricted to the cell body and dendrites
- L1, like other axonal components, is added to the axon at the growth cone
What is meant by neurons having an actin dependant diffusion barrier?
Maintains the membrane compartments in the neurones as actin filaments form a meshwork directly under the plasma membrane giving shape and resilience
Why are membrane compartments requires in neurons?
Help to maintain the axons distinctiveness and neuronal polarity
Give evidence for an actin dependant diffusion barrier in neurons?
(Winckler et al., 1999)
- Beads coated with antibodies to L1 cannot easily be dragged (using optical tweezers) across the boundary into the cell body, whereas anti-GluR1-coated beads can be dragged into dendrites from cell body. This shows that there are somatodendritic and axonal domains.
- Other proteins could not be dragged across the axon boundary but could be pulled around the soma and dendrites with ease, showing there is a barrier to diffusion at the base of the axon
- Cell surface molecules appear to be held sections by the underlying actin cytoskeleton
How can optic tweezers be used to produce quantitive data?
The use of optical tweezers can not only be used to track and move beads but can also turn it into quantitative data. This can be done by using the amount of laser movement needed to push the bead to investigate the force to which the bead is bound to the cell surface
Give evidence for how neuronal polarity occurs
Dotti et al., 1988)
- Used hippocampal neurons from the mice and cultured them. They followed how the neurons grew over time
- Initially, the cell is spherical with no projections. The actin filaments under the plasma membrane maintain a barrier to outgrowth
- This barrier is then disrupted and allows the microtubules to enter allowing formation of filopdoia
- These outgrowths are very dynamic, they then expand and form neurites (any projection from cell body).
- These neurites increase in length until one becomes longer than the others and forms a growth cone (becomes polarised)
- This extension will become the axon and may then synapse with other neurons in the culture.
How is a neurite chosen to become an axon?
The cell explores different neurites until one is chosen to be the axon. This choice is considered stochastic (random but may be underlying mechanisms that are not understood)
What happens to neurites that don't become the axon?
They mature into dendrites
What kind of microtubules are present in growth cones?
What makes microtubules dynamic?
They are tyrosinated
How are microtubules stabilised?
When an axon starts to form, acetylated microtubules accumulate. These microtubules are therefore stabilised and crosslinked
Give evidence for the importance of microtubule stabilisation in neuronal polarity
- Can artificially stabilise microtubules by adding the drug Taxol to one of the neurites. This forces the choice of neurite to become the axon
- This also suggests that there must be competition between the neurites to stabilise their microtubules and that there may be a feedback loop involved to stop the stabilisation of the microtubules on neurites not chosen
What is required to ensure that only one neurite becomes an axon?
There must be feedback loops - positive and/or negative
Give evidence for the involvement of feedback loops in neuronal polarisation?
- Chop off the axon that has been chosen
-The neurone will choose another neurite to become the axon
- This shows that there must be some sort of negative feedback loop in the axon chosen to supress the other neurites from becoming axons
How could the feedback loop involved in neuronal polarity be positive?
The positive feedback loop could act by upregulating things, that are required for axon formation, in neurites that have already started to become an axon
What are the theoretical models for negative and positive feedback loops?
- Reinforcing signal that is released from the leading edge of axon
- Diffusible inhibitor - diffuses through the rest of the cell and prevents another leading edge
- Limiting components – only enough components to form one leading edge
- Mechanical tension – to prevent formation of other leaning edges
Give evidence for the involvement of positive feedback loops in neuronal polarisation?
Overexpression of HRas or PI3K
- Results in multiple axons
- If you add a PI3 kinase inhibitor then you can block the induction of multiple axons showing that HRas activates PI3 kinase
- Also seen that activation of PI3K leads to the activation of HRas – a positive feedback loop
Give evidence for the involvement of negative feedback loops in neuronal polarisation?
HRas is depleted from non-axonal regions
- To begin with, there are no areas that have particularly high levels of HRas protein – evenly distributed
- When the cell becomes polarised, HRas becomes concentrated in the axons and a decrease of HRas in the cell body and other neurites
- This shows a negative feedback loop - a limiting factor
Give the role of PI3 kinase in microtubule stabilisation
- Downstream of many signaling receptors, PI3K elevates PIP3 which phosphorylates Akt.
- This inhibits the activation of GSK3beta.
- GSK3beta normally will destabilize microtubules
- Can see that phosphorylated Akt can be seen in the tip of the growth cone of nascent axons but not neurites
- Inhibition of GSK3beta results in the induction of multiple axons showing the importance of microtubule stabilisation
Give a protein involved in asymmetric division that affects neuronal polarity
Give the role of Par-3 in C.elegans
- Through genetic screens, mutations that affect asymmetric division were found
- There are partition defective genes that fails to develop asymmetric divisions. They are required for egg polarity after sperm entry. They Initiate polarisation through the mutually antagonistic interactions of the Par-3 complex with Par-1/2
- Downstream these affect microtubule organisation which in turn differentially localises components of the cell
Give evidence for the importance of Par kinases in neuronal polarity
Downstream to the PAR kinases are LKB1 and SAD kinases
- Loss of SAD kinase leads to the failure of acetylated tubulin to predominate over tyrosinated tubulin. Results in the loss of axons
- Loss of LKB1 kinase also leads to the failure to make axons
What are the leading and trailing processes?
Leading and trailing process refer to the direction of travel from the cell body
What is the role of LKB1?
LKB1 is required for axon initation in vivo
- LKB1 is phosphorylated by PKA. The phosphorylated form of LKB1 activates SAD kinases which affect microtubule association protein stability and axon initiation
- The phosphorylated form is present first in the trailing process and then in the axon
- There is therefore some evidence that phosphorylation only occurs when the trailing process forms
Give evidence of the importance of LKB1 in axon initiation
If LKB1 is lost in the cortex, using utero electroporation of siRNA in mice, then there is loss of axon initiation
What are the extracellular initiators of polarity?
How was the role of TGFbeta in neuronal polarity discovered?
Clinically, several genes underlying mental retardation affect cortical development, disrupting especially neuronal progenitor migration
- Milder mental retardation associated with TGFß receptor mutations
- TGFbeta is expressed in the ventricular zone of the developing cortex and can initiate axons in vitro
What are semaphorins?
Semaphorins are a family of inhibitory guidance cues
How were semaphorins identified?
They were identified by the biochemical purification of the factor from the retina that is responsible for the collapse of sensory axons. They can be membrane bound (retinal axons) or secreted (Sema3A)
What are the roles of Sema3A?
- Sema3A is expressed in a gradient from basal to apical and attracts dendrites basally
- Sema3A also promotes dendrite formation at the expense of axons in vitro
- Sema3A increases [cGMP] and suppresses [cAMP], thus inhibiting PKA phosphorylation of LKB1 and GSK3ß*
What can manipulation of cAMP and cGMP lead to?
Opposite effects on axon and dendrite formation
How do Sema3A and TGFbeta initiate neuronal polarity extracellularly?
- Apically, TGFß phosphorylates Par6 to promote axon specification
- Basally, Sema3A increases [cGMP] and suppresses [cAMP], thus inhibiting phosphorylation of LKB1 and axon formation and causes the formation of dendrites
What are lamella and filopodia?
Lamella and filopodia are made up of different kinds of F-actin. In lamella, the actin bundles are crosslinked into a net. In filopodia, the actin bundles are polarised to form larger bundles. Neither are stuck down but instead are highly motile
What is actin treadmilling?
In a resting growth cone, the actin in the filopdoia are flowing inwards from the tips as actin is being add
What happens to tubulin in the growth cone?
The tubulin is dragged sporadically into the filopodia. This happens much more dramatically when the growth cone encounters an attractive cue
How was the anatomy of the growth cone when in contact with an attractive cue investigated?
A researcher coated an attractive cue onto a bead and sew the extension of the growth cone towards the bead. The tubulin gets redirected in that direction.
The whole cytoskeleton reorganises itself upon contact with an attractive cue
What happens when the growth cone meets an attractive cue?
- F-actin treadmilling slows (is attenuated) and F-actin accumulates
- F-actin accumulation stabilises the filopodium and drags microtubules into the back of the filopodium: ‘
Explain the model of actin treadmilling when at an attractive cue
- When encountered a promoting cue, a molecular clutch (orange and purple clutch) is engaged and rearward actin treadmilling slows resulting in forward movement of the filopodium
- Actomysoin-based-actin-tubulin link (yellow microtubules) pulls microtubules into the wake of extending filopodium (where the actin was)
- See diagram
What are cadherins?
Cadherins are a type of cell adhesion molecules that are important in the formation of adherens junctions between cells. They bind homophilically between cell membranes
What cadherin is predominate in the nervous system?
How was it shown that N-cadherin binds homophilicaly?
They micro patterned a glass with islands of N-cadherin protein. They then put neurones down onto the substrate that have GFP N-cadherin fusions so that N-cadherin can be seen inside the cell. Can see accumulation of GFP N-cadherin where there is N cadherin on the substrate. This shows that N-cadherin binds homophilically
Give evidence for the molecular clutch
N-Cadherin experiment - traced actin molecules in the cell
- Can see actin treadmilling but not in all actin molecules
- Actin molecules that aren’t treadmilling are over the N-cadherin islands
- Can then plot the motion of these particles to see where actin molecules spend more time. This shows that the actin molecules spend more time over the N-cadherin then over other molecules suggesting there is something that arrests actin flow. This provides some evidence for a molecular clutch
What are the two theories about how the molecular clutch works based on the N-cadherin experiment?
- A cell surface receptor (in this case N-cadherin) can bind to a ligand (N-cadherin substrate) and engage the clutch that arrests the actin flow. This is likely to be due to a direct interaction between something on the intracellular domain of the axon and the cytoskeleton.
- Binding of the receptor on the surface to the substrate generates a signal which activates intracellular crosslinking of F actin by second messengers. This would stop any backwards flow and force a forward flow. The clutch may not involved direct interaction
What happens to the F-actin in the growth cone upon contact with semaphorins?
F-actin decreases and the growth cone collapses. This is opposite to what occurs when there are positive cues (F-actin accumulates)
What are Rho GTPases?
Rho GTPases are important regulators of the actin cytoskeleton. When they are bound to GDP they are ‘off’ but when phosphorylated to the GTP bound state, they are in the ‘on’ state
What regulates Rho GTPases?
Their activity is regulated by GAPs (GTPase activating protein) which deactivate Rho GTPase and GEFs (Guanine nucleotide exchange factor) which activate them as they add a phosphate
What affect do Rho GTPases have an the actin filaments in fibroblasts?
Three well-studied members of the Rho GTPase family promote the formation of specific actin-based structures when constitutively activated in fibroblasts:
- RhoA induces stress fibers
- Rac induces the lamellipodia
- Cdc42 induces the filopodia
By contrast, dominant negative (DN-) versions suppress the formation of these structures
What affect do Rho GTPases have on axon growth?
When activated (“on” state), Rac & Cdc42 seem to be positive regulators of axon growth, whereas activated RhoA is a negative regulator of axon growth
The balance of these decide whether an axon is going to grow
What is required for the collapse of a growth cone?
RhoA is required for a negative cue (e.g. Sema) to collapse a growth cone
What does constitutively active and dominant negative forms of RhoA lead to?
Constitutively active RhoA causes neurite retraction, whereas dominant negative RhoA blocks collapse response
What does constitutively active and dominant negative forms of Rac and Cdc42 lead to?
They both block axon growth
Why do constitutively active and dominant negative forms of Rac and Cdc42 block axon growth?
Growth requires assembly and disassembly of actin structures
This can also be seen during axon guidance
The collision of two attractive growth cones causes the actin cytoskeleton to dissemble in one area and reassemble in another
If f-actin can’t be disassembled then direction of growth cannot be changed
This therefore means that both dominant negative and constitutively active Rac will block axon growth
Are Rho GTPases permissive or instructive for axon growth?
Evidence suggests that they are instructive, as factors that collapse growth cones activate RhoA and downregulate Rac
How are Rho GTPases regulated to guide axons?
Several guidance factor receptors either bind to modulate Rho GTPases directly or bind to GEFs or GAPS which regulate the Rho GTPases
Give an example of how semaphoring signalling guides axons
Signalling alters RhoA/Rac balance
- Normally in drosophila, semaphorins are expressed in certain muscles, but not others, to guide innervation by motor neurons. The motor neurons will therefore innervate muscles that are not expressing semaphorins
Changing relative levels of Plexin B, Rac or RhoA changes the sensitivity of motor axons to the Semas
- Increasing plexin, makes the neurones more sensitive to Semas. If increase this more, then will become hyposensitive
• Less Rac, more sensitive. Increasing Rac in the neurons makes them less sensitive
• Decreasing RhoA makes it less sensitive
What are plexins?
Receptors for semaphorins
Give the model for sema signalling
- With no semaphorins, plexin receptors are not activated. The levels of free Rac outweigh free RhoA causing axon growth. When sema binds to plexin it causes sequestration of Rac-GTP. This causes the balance of RhoA to increase over RacA preventing axon growth
- Balance between these factors determines the sensitivity to semaphorins
Explain how regulating Rho GTPases by GEFs will lead to greater specificity
- The Rho GTPase family is relatively small and members are broadly expressed (not tissue specific)
- GEF and GAP families are much larger, and more restricted in their expression
- Suggests that regulation via GEFs and GAPs of these would afford much greater specificity
Give an example of how regulating Rho GTPases by GEFs can guide axons
Ephrins, inhibitory cues, signal via GEF called Ephexin which simultaneously regulates RhoA, Rac and Cdc42. This again shows the importance of the RhoA/Rac balance
Give evidence for the ability Ca2+ to turn growth cones in vivo
- Added a calcium indicator to growth cones in vivo. The growth cones are seen to have high levels of calcium
- Also investigated using light activated Ca2+ cage
What is a light activated Ca2+ cage?
Molecules, such as EGTA, that when light is flashed it releases the calcium that is bound
Give evidence for the ability Ca2+ to turn growth cones in vitro
- In vitro, addition of netrin stimulates the accumulation of intracellular calcium
- Ryanodine releases calcium from intracellular stores. f add ryanodine in vitro, then growth cones turn towards it. Ryanodine activates Rac/cdc43 and supresses RhoA
What are the key ways to regulate actin cytoskeleton through Rho GTPases?
- Control filament disassembly – by Cofilin
- Regulate branching – initiated by Arp2/3 in response to activation by WASp proteins
- Termination of branch extension by capping proteins
- Filament assembly – regulated by Prolifin and Thymosin
- Actomysoin contractility – essentially crosslinking between branches structures
Are microtubules or actin the first to respond to a cue?
Why are microtubules the first to respond to a cue?
Need to bring important factors for signal transduction and membrane protrusion to the site of the contact.
- Monomeric actin is imported by invading microtubules which could be the reason why actin cannot act before microtubules
- Other factors are also imported such as Collapsin Response Mediator Protein (CRMP). This promotes microtubule polymerisation
What are netrins?
Netrins are secreted proteins like laminin which can associate with the extracellular matrix and can turn commissural axons
- They act as chemoattractant proteins
Where are netrins secreted from?
Secreted from the floor plate
How were netrins discovered?
Their identity was discovered by biochemical purification which led to cloning of the gene encoding the floor plate chemoattractant protein, which is expressed along the midline of the vertebrate nervous system
What is repsonsible for turning commissural axons?
Netrins from floor pate - attracts
BMPs from roof plate - repeals
What was the hypothesis for Lyuksyutiva et al, 2003?
This paper explored the idea that there was a gradient that determined that most the axons that crossed the floor plate then turned anteriorly. They had two theories for how this happened:
- There was an anterior-posterior gradient of either an attractant making them turn anteriorly or a repellent that drives them anteriorly. This would be a long rage diffusible cue
- The same but the cues were short range non-diffusible (e.g. gene expression gradient)
How did Lyuksyutiva et al, 2003 show that the molecule was diffusible?
- If took a small piece of spinal cord and put in a collagen gel, then a diffusible molecule should diffuse out of the explant into the collagen so should therefore see a disruption to the axon guidance
What methods did Lyuksyutiva et al 2003 use to show the molecule acted over a long rage?
Take out spinal cord before the axons begin to extend and open it out to lie it flat on the gel. Leave it for a few days and allow the axons to grow. Fix the cells with paraformaldehyde and a lipophilic dye. Some of the axons have turned
How did Lyuksyutiva et al 2003 experiment show that the molecule was an attractant?
They took larger explants and put in the middle of AP axis and found that there is an attractant at the anterior end
- If it was a repellent and explants were put in culture for a given amount of time, the gradient will push axons in one way. After some time has passed, the gradient will have diffused so that both ends are now low and the highest concentration of repellent is in the middle.
- This would mean that at the posterior end of the explant the axons will turn down the gradient but at the anterior end it would be unaffected.
- If it was an attractant, the opposite is true meaning that the anterior end would turn down the gradient.
- The diffusible molecule is therefore an attractant
What evidence did Lyuksyutiva et al 2003 have to show that Wnt4 can turn commissural axons and is expressed in a gradient?
- If they took cells and transfected into it a control or a vector with Wnt4 (forced Wnt4 expression) and placed them at the anterior or posterior end of a small explant, they could dictate the way the axons can turn
- Frizzled3 knockout mice have confused turning after the floor plate showing Wnt is a key guiding molecule
How long of gradient can Wnt act over?
Long gradients (4mm)
Why are Wnts, BMPs and Shh very important in development?
Wnts, BMPs and Shh are also used earlier to pattern the spinal cord
Shh works redundantly to guide commissural axons
Factors that are used early to pattern neural progenitors are then used later in development.
What are the two ways cells use to measure gradients?
Temporal detection – change of concentration over time. Cell must compare amount of ligand at two different time points
Spatial detection - change of concentration across the cell. Cell must compare the amount of ligand at two different points in its surface
What type of organisms use temporal and spatial detection of gradients?
Spatial detection is the most important in eukaryotic cells because they are slow moving. However, bacteria move quickly so temporal detection is used more
What models are used to study gradient detection?
Mostly carried out in neutrophils and leukocytes or in the slime mould Dictyostelium
Investigate using their chemotaxis
When is the accuracy of growth cone orientation most accurate?
The accuracy of orientation towards a source depends on the absolute concentration of the chemoattractant. It is most accurate when closest to the dissociation constant (Kd) of the receptor
When do leukocytes orient their axons with accuracy and how can this be shown?
When the gradient is less than 0.2%
- Gradient is expressed as % change per micron
- A graph plotted shows that orientation towards the source is only 50% (due to chance) when the chemoattractant gradient is 0 but when it starts to increase to 0.2 it reaches 100%
What is required from growth cones to detect gradients?
Require very small differences in receptor occupancy to be detected across the cone
How can growth cones enhance there ability to detect gradients?
Amplification and adaptation
What is amplification in terms of enhancing ability to detect gradients and what is its suggested mechanism?
- Amplification requires local enhancement of signal together with the inhibition of signal reception in other parts of the cell
- One suggested mechanism involves clustering of receptors and/or signalling components, in regions where receptors are activated, by transporting components from other parts of the cell
Give evidence (4 pieces) for amplification as a way of enhancing growth cones ability to detect gradients?
Chemotaxin Dictyostelium (Aubry et al, 1999)
- Chemoattractants in leukocytes and Dictyostelium activate PI3 kinase via a GPCR
- Can detect PI3 as bound to proteins with Ph domains - fuse GFP
- The chemoattractant is pipetted to the cells. The PH domains are orientated towards the chemoattractant
This can also be seen in neurones (Ming et al, 1999)
- PI3K inhibitors disrupt growth cone turning induced by netrin
Localisation of PH domain proteins during growth cone stimulation
- Xenopus spinal cord neuron expressing Ak1-PH-GFP
- Add BDNF and the PH domain in the filopdoia
Asymmetric vesicle transport in growth cones
- Vesicles are transported towards point of contact with attractive cue
What is adaption in terms of enhancing ability to detect gradients?
If growth cones operate most discriminately when the chemoattractant concentration is closest to the receptor dissociation constant, then receptors will saturate as concentration increases
Signal saturation leads to desensitisation and resensitisation
Give evidence for adaptation in terms of enhancing ability to detect gradients?
My-Ming Poo carried out a xenopus spinal cord neuron turning assay
- Axons of isolated neurons from early embryonic Xenopus spinal cord respond to netrin pulsed into a dish from a pipette. Growth cone does not turn towards inactive netrin (control)
- Pulsed netrin to form a gradient – checked using a fluorescent dye
- Look at growth cone responses over time: over about 20 mins, the growth cone starts to turn towards it but 20 minutes after that, the growth cone starts to turn away
- The growth cone takes a wiggling pathway to get towards the pipette. This could be due to desensitisation and resensitisation
- However, it could also be due to the netrin gradient being set up by a pulse
How did My-Ming Poo ensure that the growth cones wiggling pathway was due to desensitisation and resensitisation?
They added netrin to the bath before they put the pipette in the bath. The growth cone that has already been exposed to low levels of netrin doesn’t turn immediately but does eventually. This is good evidence that growth cones desensitise and resensitise
Does adaptation also occur with repellants as well as attractants?
Piper et al, 2005
Used Sema3A (repellant) and saw similar adaption
What happens to axons after they cross the midline?
- Lose responsiveness to netrins
- Become sensitive to repellants
Give an experiment showing how axons lose responsiveness to netrins
Shirasaki et al 98
- Experiment in rodent hindbrain. Added an ectopic floor plate on one side of the hindbrain
- Axons exposed to an ectopic floor plate before reaching midline respond by turning
- Axons exposed to ectopic floor plate only after crossing midline no longer respond
- Sensitivity of axons to floor plate (netrins) changes after midline is crossed
- Explains why, in the hindbrain, commissural axons can continue past floor plate without turning
Give an experiment showing how axons become sensitive to repellants
Zou et al 2000
- After crossing the midline, commissural axons become sensitive to something inhibitory in the floor plate
- Initially these axons must be sensitive to netrin, but not sensitive to the inhibitors. Once across the floor plate, the sensitivities must switch
Why don’t axons in the spinal cord go straight on after crossing the midline like those of the hindbrain?
The inhibitory molecules in the floor plate are semaphorins and proteins called slits
As well as being expressed in the floor plate, these are also expressed in the ventral spinal cord thus creating a channel through which commissural axons grow
What molecules are involved in midline crossing in the flies?
Insect midline glial cells express diffusible attractants (netrins) and cell surface repellants (slits)
What is the phenotype of a roundabout mutant?
The axons continue to recross the midline and go around in circle
What is the phenotype of a commissureless mutant?
The axons never cross the midline
What is Robo?
Encodes a receptor for the inhibitory protein Slit
Where is Robo expressed in flies?
Robo protein is expressed at high levels on axons that don’t cross the midline
Commissural axons initially express low levels of Robo protein, but high levels after they cross
Where is Comm expressed?
in midline cells, and in those neurons, that normally cross the midline
What happens when the expression of Comm is formed everywhere?
Robo protein is lost everywhere resulting in a phenotype just like the Robo mutant
What is the role of Comm?
What are the two models of how Comm regulates Robo?
- The presence of Comm forces sorting of newly synthesised Robo into late endosomes
- Homophillic binding of comm (between Comm on the growth cone and on the cells on the membrane) triggers clearance of Robo from the cell surface. Once the axons had crossed the midline, Robo was able to reach the surface, leading to a repulsive response
Who tested the two models of how Comm regulates Robo?
Keleman et al 2005
How did Keleman et al 2005 test if Comm is required in midline cells for correct crossing?
- They used a neuron-specific promoter to drive axon marker in a subset of crossing neurons. The marker behaves like most of the axons
- They used the same promotor to also drive Comm expression in the neurons in a Comm mutant. They found that they could rescue midline crossing
- The rescue is at least as good as when Comm is also turned on in midline cells (as well as in the neurons)
- Don’t need Comm midline expression to rescue
- Disagrees with the clearance model (homophillic binding not occurring)
How did Keleman et al 2005 test if Robo is prevented from going to axons in the presence of Comm?
- They tested this by visualising Robo protein in axons in the living embryo using Robo-GFP fusion
- Robo-GFP in the axon is not expressing comm. Robo is being transported along the axon from the cell body to the growth cone
- Then drove expression of Comm in an embryo which expressed Robo-GFP. They found that Comm stopped the transport down the axon, restricting it to the cell body
- Tells us that Comm is responsible for controlling transport of Robo into the axon
What is the main difference between the sorting and clearance models for the function of Comm?
In the sorting model - Robo should not be shipped down the axon in the presence of Comm, whereas it should if instead it is cleared from the cell surface
Is the sorting or clearance model correct?
Sorting model appears correct
- Still don’t understand why Comm is on the midline or what controls the upregulation of Robo on contralateral side
Why is loss of response to netrins in vertebrates likely to involve contact with the midline?
- This is thought to have been evolutionary set up like this because Robo protein is made before axons ross, It can rapidly appear on the cell surface when the midline is encountered
Give examples of cell surface proteins that change expression in commissural axons cross the midline in mammals
TAG-1 and L1
Explain the expression of Robo in commissural axons in mammals?
Robo is expressed at low levels before crossing and high after crossing. This correlates with the increase of sensitivity of post-crossing axons
Is there a Comm homolog in vertebrates?
What are three robots-like receptors in mammals?
Robo 1 and 2 and Rig-1
Explain the expression of the three Robo like receptors in commissural axons in mammals?
Robo 1 and 2 are expressed at low levels until the axons cross the midline. Rig-1 is expressed at high levels until the axons cross the midline
What is the phenotype of a Rig-1 knockout?
Prevents midline crossing by commissural axons. This is surprising because knocking out Robo in the flies, resulted in more midline crossing as Robo is the receptor for slit
What are the theories that explain Rig-1 knockout phenotype?
- Rig-1 is an attractive receptor required for floor plate crossing. Therefore, would not cross the midline in a Rig-1 knockout
- Rig-1 prevents premature sensitivity to a floor plate repellent
Give evidence for Rig-1 preventing premature sensitivity to a floor plate repellent
Sabatier et al, 2004
- Carried out a floor plate assay and can see axons extending
- In a Rig-1 knockout mice, it appeared that the axons were not attracted to the floor plate. This therefore sounds like the first model is correct.
- However, they blocked slit function in a Rig-1 knockout using soluble Robo, then the axons were attracted to the midline
- To investigate this more, they added slit cells
- Wild-type pre-crossing commissural axons ignore Slit as Netrin has also been added whereas those lacking Rig-1 are repellent
- Therefore, function of Rig-1 is to prevent premature Slit sensitivity
What is the role of netrin in controlling slit?
Netrin overrides the action if slit but only if Rig-1 is present
What molecule in flies is the function of Rig-1 analogous to?
- both prevent Robo from signalling before the midline
Give an experiment that shows netrin response is lost?
- Isolated neurons from embryonic xenopus spinal cord respond to netrin in axon turning assay
- The effect of netrin is negated when slit is also present. These neurons are thought to be equivalent to post crossing neurons. When add both slit and netrin together to these neurons, the positive response to netrin is lost
- This is specific to netrin since turning induced by another molecules (DBNF) that binds to a different receptor is not affected by slit
What are the two models that explain why netrin response is lost after crossing the midline?
- Ligand-ligand interaction: Slit stops netrin binding to its receptor by itself binding to the netrin ligand
- Receptor-mediated silencing: Slit binding to its own receptor, Robo, may somehow silence netrin receptor, DCC
Give an experiment that tested which model was correct when explaining why netrin response is lost?
- In the xenopus, you can introduce new forms of the receptors by injecting RNA into one cell embryo, letting it develop and then culture neurons from its spinal cord.
- They therefore expressed hybrid versions of Robo: the intracellular domain of Robo with the extracellular domain from another receptor (TrkA)
- Now find that NGF (ligand for TrkA) can block the netrin turning response.
- Since NGF does not do this normally, this argues against model 1 and in favour of model 2
- These and other results indicate that the silencing of the netrin response by slit is due to an interaction between the cytoplasmic domains of DCC and Robo.
Give an overview of what happens when commissural axons cross the midline?
- On pre-crossing axons, Rig-1 and small amounts of Robo1 and 2 are present on the cells surface. Slits are present in the environment but they are not responded to because Rig-1 is inhibiting Robo1 and 2. Instead, the axons are responding to netrins which attract the axons towards the floor plate by binding to its receptor DCC
- Once the axons cross the floor plate, Rig-1 somehow disappears meaning Robo can bind slit can generate a signal which silences the DCC receptor stopping the response to netrin and repulses axons
What causes commissural axons to become sensitive to repellents after crossing the midline?
- Something could happen when the axon touches the floor plate
- Or there could just be a time switch- as the axons mature
Give evidence for axons touching the midline leading to sensitivity to repellents?
Stoeckli et al 1995
- Contact with the floor plate affects responsiveness
- TAG-1 is an adhesion molecule expressed on commissural axons that binds to another adhesion molecule, NrCAM, found on the floor plate
- Added antibodies to either molecule during the period the axons were crossing the midline. This lead to axons turning on the wrong side
- Adding NrCAM to commissural axons before they cross the midline was enough to stabilised Sema receptor PlexA1
- There is a degradation mechanism that degrades PlexA1 on axons before they get to the midline but contact with NrCAM at the midline stops this degradation, allowing PlexA1 to reach the cell surface and respond to semas
What is PlexA1?
Receptor for semas
What are the two different types of gradients that can be set up?
- A gradient set up by diffusion from a point source (such as that used by Ming et al) - typically an exponential gradient - has a theoretical maximum range of 1mm, which corresponds well to supposed point source ranges in vivo
- By contrast, a substrate bound gradient, typically more linear in shape, has a theoretical maximum range of close to 1cm.
- Again, this corresponds well with what is observed in vivo
What to point source gradients depend on?
A limited diffusion rate.
Some mechanism for removing the guiding molecule - a ‘sink’ - otherwise gradient will flatten with time (depending on diffusion rates)
Give evidence against point sources in vivo?
Netrin mRNA is more complex
- Netrin 1 in chick is localised to floor plate
- But netrin 2 is expressed at low levels throughout (Kennedy et al. 1994)
- The laminin-like structure of netrin suggests it may be bound into the ECM
- Recent evidence even suggests that netrin is not required in the floor plate for Commissural axon guidance: Morales 2017
- Suggests that netrin may be bound to the extracellular matrix rather than diffusible
Wnt expression is graded, not point source
- This expression will vary temporally. Expression is likely to change from rostral to caudal as development proceeds
Give evidence for semas being switch on by Shh
- Add netrin to sema expressing cells and allow ougrowth from the explant
- When only netrin in the dish the axons are not repelled by the sema
- Add Shh, the axons are repelled away from the sema despite netrin being present
Give evidence for Shh guiding commissural axons to floor plate
- Cyclopamine blocks Shh and stops the attraction
- Knocking out smoothened in commissural axons lead to mistakes in the pathway
Give evidence for commissural axons becoming sensitive to repellents being because of a time switch
Switch is also cell intrinsic
- Add commissural axons at an early stage before they have reached the floor plate and culture them. TAG1 disappears and L1 appears over time
- This isn't floor plate dependant and shows that changes just occur as axons mature
How does cell intrinsic responses lead to a change in response to Shh?
The initial response of commissural axons to Shh in vitro is attraction but then turns to repulsion after 2-4 days
What controls the change in response to Shh?
What is 14-3-3?
A par like protein
Give evidence for the role of 14-3-3 in changing axons response to Shh?
- If block its expression with shRNA. Maintain the attraction to Shh
- 14-3-3 is increased in the post crossing axons – showing its involvement in Shh responsiveness
- Overexpression of 14-3-3 can lead to premature tuning of axons
What other molecule does 14-3-3 effect?
Wnt – mutual inhibition between Wnt and Shh to drive the growth cones in their pathways
What are the roles of Shh in axon guidance?
Used as an attractant to the floor plate, involved in its switch to a repellent and then in AP guidance
How does Shh guide axons along the AP axis?
Makes the axons turn in an anterior direction due to it becoming repellent
What happens in a loss of function Shh in commissural axons?
Failure of attraction in vitro. Also fails to be repelled – axons wonder
How are responses to cues modified?
Levels of intracellular cyclic nucleotides are critically important in determining the polarity of a growth cone’s response to chemotropic molecules
What is meant by polarity in terms of cues?
Polarity in this case means whether the response to a cue will be attraction or repulsion
How can the response of many guidance cues be reversed?
By the combination of receptors that are present which therefore manipulating the concentrations of intracellular cyclic nucleotides
- If just DCC present then cAMP levels will increase so attractive but if DCC is accompanied by co receptor Unc5 then it is repulsive because cAMP decrease
What happens to commissural axons if block PKA?
If block cAMP-dependent protein kinase A (PKA), growth cones turn away from the netrin source
The effect is all or nothing suggesting cAMP acts as a switch determining the direction of the netrin response
The turning is calcium dependent irrespective of which direction
What repulsive cues have their effect by inhibiting cAMP?
Where in the growth cone are high concentrations of Ca2+ seen?
On the side of the growth cone facing the source of the cue, regardless whether the cue is attractive or repulsive
What occurs, in respect to Ca2+ flux, when an attractive cue is present?
- Attractive cues elevate cAMP and either activate TRPC channels at the cell surface to let in extracellular Ca2+, or elevate intracellular IP3
- Both Ca2+ and IP3 trigger further release of Ca2+ from intracellular stores
- Store release of Ca2+ feeds back to further elevate cAMP, which in turn causes further increase in Ca2+.
- This positive feedback loop results in a high amplitude Ca2+ flux which activates extension
What occurs, in respect to Ca2+ flux, when a repulsive cue is present?
- Repulsive cues elevate cGMP and activate TRPC channels at the cell surface to let in extracellular Ca2+
- cGMP, however inhibits Ca2+ release from intracellular stores (as well as from L-type CCs at the cell surface). It also inhibits rises in cAMP
- Therefore, store release of Ca2+ does not occur and cAMP levels do not get amplified
- This low amplitude Ca2+ flux then somehow activates repulsion and collapse
Why does an increase in Ca2+ cause axons to move towards a positive cue?
When there is a positive cue, you get transport of vesicles towards where the positive cue is and get membrane insertion to cause movement. This happens in response to the high Ca2+
Why does a decrease in Ca2+ cause axons to move away from a repulsive cue?
When there is a repulsive cue, we get an increase in clathrin mediated endocytosis. This leads to removal of the membrane away from the cue
This is due to a decrease in Ca2+
Give example of other molecules that can modulate polarity of the response to cues
- Integrins bind to laminin which lowers cAMP levels
- NO can inhibit cGMP
Give an example of the environment changing the responses to cues
In the tectum
- Retinal ganglion cells are attracted to netrin that is expressed by cells in the optic nerve head
- Contact with laminin in the optic nerve reverse the response to netrin and may serve to direct axons away from the optic nerve head
- This is a real example of the environment changing the responses
What molecules are known to inhibit CNS regeneration in mammals?
MAG and Nogo
- Found in CNS myelin
How do these molecules inhibit CNS regeneration in mammals?
They effect cAMP levels by activating RhoA
Where can regeneration occur?
Regeneration after axon injury occurs in lower vertebrates, the embryonic CNS and in the adult mammalian PNS, but not in the adult mammalian CNS
Why can't the adult CNS regenerate?
- Failure to activate growth promoting program in injured neuron
- Presence of inhibitory factors in CNS myelin (MAG, Nogo)
- Formation of ‘Glial Scar’ that presents a physical barrier to axon growth
Can CNS axons ever regenerate?
Yes if given the appropriate substrate and if the right genes are activated
Give evidence for the ability of CNS axons to regenerate
Experiment in spinal cord – conditioning lesion
- The dorsal root ganglia have an axon that go to the periphery (can regenerate) and also have centrally projection (can’t regenerate)
- They did a conditioning lesion, cut the peripheral branch first and then the central
- Both branches then regenerated
How do all MAG/Nogo/Omgp inhibit CNS regeneration?
They bind to NgR
Activation of NgR changes the RhoA/Rac balance (activates RhoA) leading to growth cone collapse
How does increasing cAMP levels give some therapeutic opportunities for CNS regeneration?
Elevating cAMP levels activates PKA which in turn phosphorylates RhoA leading to its inactivation
What is Db-cAMP?
Membrane soluble version of cyclic AMP
Give evidence that Db-cAMP can promote regrowth of CNS?
- Db-cAMP can allow growth of sensory fibres in vitro in the presence of CNS myelin
- Finally show that db-cAMP injection into the DRG prior to lesion (cf pre-conditioning lesion) can enhance regrowth through subsequent dorsal column lesion
- This isn’t therapeutically useful as would have to know when the damage occurs but shows that is possible
Why is the addition of Db-cAMP alone not enough?
Because there is no functional recovery of regrown neurons
What are regeneration associated genes (RAGs)?
Transcriptomics (analyzed which genes are turned on) reveals large arrays of changes after the conditioning lesion and RAGs were turned on
Give evidence for Rho-kinase inhibition promotes CNS regrowth
Fournier et al
- They tested the regrowth in the dorsal column monitoring regrowth in the corticospinal tract
- Found that adding C3 transferase (a RhoA inhibitor) effects regrowth in cultured neurons but they couldn’t make it work in vivo
- They thought this was because of a delivery problem of the enzymes
- However, a different ROCK inhibitor (y27632) administered to the same lesion does promote growth through the dorsal column lesion
Name a drug for CNS repair involved in Rho-Kinase inhibition
- It is a cell permeable version of C3 transferase
- Works in animals and in humans
- It is currently entering phase 2 clinical trials
Give evidence for the role of NSAIDs in CNS regeneration
Fu et al (2007)
- Found similar results as RhoA inhibitors using ibuprofen
- Enhances DRG growth on myelin
- Inhibits RhoA activation in injury site. This is shown that in the control there is increased RhoA activity after injury but with ibuprofen this doesn’t happen
- They showed that there was enhanced recovery to the distal spinal cord in a similar lesion as the stride length of mice increased
How is ibuprofen though to enhance CNS regeneration?
This is not a function of the COX inhibitory power of ibuprofen because other NSAIDs don’t work. It instead acts by activating a transcription factor which upregulates a phosphatase which may inhibit a RhoA GEF to supress RhoA activation
Give a study into CNS regeneration not involving RhoA
Park et al (2008)
- They reasoned that pathways in regulating cell growth may also regulate the ability of axons to grow
- They tested a pool of mice carrying conditional knockouts in major growth control genes including: retinoblastoma (Rb), P53, Smad4 for the ability to regrow optic nerve after injury
- Mice carried conditional mutations of each of the different genes
- They also added GFP to allow them to see the axons which have had the gene knocked out
- In the control, growth stops at the site of the injury after 14 days. The only gene that affected regrowth was knocking out PTEN
How was the conditional knockout made in Park et al 2008?
Mice carried conditional mutations of each of the different genes and added an adenovirus to the mouse eye which is carrying gene in which Cre is being expressed under the control of a Strong ubiquitous promotor. This would cause the knockout of that gene.
How did PTEN effect CNS regeneration?
It regulates the mTor pathway
PTEN therefore blocks the downregulation of this pathway when injuries occur allowing for some regrowth
What happens to the mTor pathway normally in response to CNS damage?
The mTor pathway is progressively inhibited as development ceases in normal animals. In injured adults, there is more downregulation of the pathway
How did they show that it was that branch of the mTor pathway that blocked CNS regeneration?
The showed that it is this branch of the mTor pathway by knocking out TSC1, part of the mTor pathway. It resulted in a similar phenotype to the PTEN knockout
Give a study into PTEN deletion enhancing spinal axon regrowth?
Liu et al, 2010
- Looked at the corticospinal tract and some regenerating neurons could form new synapses. However, there was no functional recovery
- The theory is still promising
- They are therefore looking for a drug that inhibits PTEN – usually trying to upregulate it as it inhibits cell growth in cancers
What is the tectum?
Explain the topographic mapping of the retina?
The nasal (anterior) axons from the retinal ganglia cells synapse onto the posterior of the tectum whereas the temporal (posterior) axons synapse onto the anterior tectum.
Give evidence for the topographic organisation of the tectum
Stripe assay (Walter et al, 1987)
- He laid out the tectum with stripes from the anterior and the posterior ends laying adjacent
- Nasal axons could grow over either anterior or posterior parts of the tectum whereas temporal axons could only grow over anterior and avoided the posterior membranes
- This appears to be due to temporal axons avoiding a repellent factor in the posterior stripes as posterior membranes cause temporal growth cones to collapse in vitro
What is the repulsive factor present in the posterior membranes of the tectum?
Two Ephrins (A2 and A5) which are expressed in an anterior (low) to posterior (high) gradient
How do the Ephrins inhibit temporal axons from growing in the posterior but no nasal
Ephrins are expressed in a gradient in the tectum and superior colliculus and the receptors for these two Ephrins are expressed in a counter gradient in the retina. Temporal (high) and nasal (low)
What occurs in Ephrin A2 and A5 knockout mice?
Temporal neurones project their axons into the posterior tectum and the topographic map is disordered. This is consistent in the idea that Ephrins are important in topographic mapping
How does the topographic organisation of the retina differ in rodents then in chicks?
In rodents, both temporal and nasal axons growth all the way to the posterior end of the tectum. Subsequently, there is branching of the axons which causes synapsing. It is this process that is sensitive to the Ephrins
How is refinement thought to occur in the rodent?
There appears to be competition between the axons for synaptic partners – involving electrical activity so that weaker synapses get eliminated causing the temporal axons to be pushed back to where we see them in the adult animal
Give a study showing that electrical activity can modulate responses to guidance cues
Ming et al, 2001
- They found that electrical stimulation can reverse or enhance guidance cues
- High concentrations of netrin leads to axon turning in vitro but not low concentrations. However, if add electrical stimulation to the growth cone when adding low concentrations of netrin, it enhances the response
- When there is no electrical stimulation MAG repels axons in vitro. The addition of electrical stimulation causes MAG to act as an attractant
What does electrical activity modulate to cause a change in response to guidance cues?
Ca2+ and cAMP
Give evidence that it is Ca2+ and cAMP that causes a change in response to guidance cues?
- Neuron has been loaded with a calcium indicator dye. Turn on electrical stimulation then it leads to elevation of Ca2+. This is dependent on the electrical activity level
- Electrical activity can reverse MAG to an attractant. Blocking cyclic nucleotides can reverse the response to MAG once again
- Electrical stimulation turns up the level of cAMP in a calcium dependant way. The more electrical activity the more stable the Ca2+ elevation
What can chelating agents be used for?
To detect Ca2+ or uncaging of calcium
How are chelating agents used to uncage calcium?
Uncaging breaks the bond of EDTA and releases the calcium that is bound
How can chelating agents be used to detect calcium?
EDTA like molecules can also be attached to fluorophores. When calcium is bound, it changes the way they interact meaning it illuminates stronger when Ca2+ is bound
Give an example of refinement being activity dependant
This can be seen in the formation of ocular dominance columns in the visual cortex
What is the hebbian rule and how does it relate to refinement of axons?
The mechanism of synapse elimination involves the localised release of neurotrophic factors which are thought to be enhanced when to cells fire at the same time
- This implies that refinement is activity dependant and relies on competition between axons
Give evidence against refinement being due to activity?
However, In the retinal tectum system, this refinement occurs in the embryo before the organism has encountered any light
How does refinement occur in the embryonic retina when there has been no light encountered?
- The embryonic retina becomes spontaneously active
- This can be seen in calcium imaging. There are waves of calcium spontaneously emerging in the embryo and spreading across the retina. These are activity dependant waves that can be blocked using activity blockers
- Even before the retina sees light, there is activity present
- Treatment with a channel blocker, blocks map refinement
Give evidence for refinement being activity dependant?
In wild type mice, the branches are pruned back by postnatal day 8. However, in a mouse that lacks b2 subunit of nAChR then the branches are not refined
What information does the eph-ephrin system provide?
It is possible that the eph-ephrin system is only providing positional information and not guiding information
- Sperry described this as latitude and longitude coordinates
- The axons could be aware of where they are in the map due to this
- The axons only extend branches at certain points depending on the signal they recieve
How does the eph-ephrin system induce branching of temporal and nasal axons?
- High levels of EphA4 in the temporal retinal ganglia, growing into the tectum. The high density of the receptor on temporal growth cones ensures enough signal to induce branching even when the ligand is at low density
- However, for nasal growth cones, the lower density of receptor on their surface means that only the ligand density is higher will enough signal be generated
Give an experiment that explains how ephrins control branching of axons?
In vitro assays
- Took panels from the retina (different concentrations of ephs in each panel due to the gradient) and looked how these different panels respond to concentrations of Ephrins
- At low concentrations of ephrins, branching is promoted
- At high concentrations, there is complete inhibition of outgrowth. This is a surprising result
- However, it does still show that there is a response to the concentration of ephs receptors present in the retina as at high concentrations of ephs complete branching inhibition occurs earlier with high concentrations of Ephrins then when there was a low concentration of ephs
What is the role of BDNF in axon branching?
Promotes axon branching but only on regions where Eph-ephrin interactions are low
Give evidence for the role BDNF in axon branching
- branching only occurred in stripes where the ephrins concentrations are low
What cause BDNF upregulation in the retina?
Enhanced retinal electrical activity results in BDNF upregulation in the environment
How do ephrins and Tkrs interact?
Physically in a neurotrophin-dependant manner
What happens after BDNF is unregulated?
- BDNF is activated
- At intermediated concentrations of ephrins, there are high levels of branching induced
- As the eph-ephrin interactions increase (either by increase in ephrins in environment or axon has lots of ephs), there is inhibition of branching occurring
- There is interaction between the electrical activity and the cues in the environment
In mammals where else is topographic mapping used in the visual system?
Guidance to retinal ganglion cells to the different layers in the LGN is also controlled by an ephrin gradient
Where else is topographic mapping used in the brain?
- Topographic mapping is used in somatosensory maps. The order is maintained topologically from sensory information to the somatosensory cortex in the brain. Posterior to anterior mapping
- When you look into the somatosensory cortex when the axons are coming from the thalamus, you can see that there is also a counter gradient of ephs-ephrins
What transcription factor is involved in setting up the ephori gradients?
- high at rostral and low at caudal
Give evidence for the role of EMX2?
In mice that overexpress EMX2, distorts the map in the gradient. Changes the visual and somatosensory cortex locations
What is the role of FGF8 is topographic mapping of the cortex?
- FGF8 is usually expressed at high levels in the top of cortex
- If change levels then so does the mapping system
- Can cause duplication of the axis by adding another FGF source to the bottom of the cortex
Give an example of a non spatial sense
What detects odours?
What kind of receptors are olfactory receptors?
How many olfactory receptors are there in humans?
- even more in animals where smelling is important e.g. dogs
How many different olfactory receptors are expressed in any one neuron?
Only one type
Are olfactory receptors organised?
- Receptors are not grouped in the olfactory epithelium, they are scattered
- It becomes organised in the sense that all the neurons expressing the same receptor go to the same place in the olfactory bulb
How was mapping from the olfactory epithelium to the olfactory bulb investigated?
Taken one olfactory gene and knocked in a marker to visualise with a blue dye.
- One particular olfactory receptor gene (P2) drives the expression of the marker and has an IRES region which allows the re-entry of the ribosome to allow translation
- Can see that p2 is scattered across the olfactory epithelium and they all converge on a particular glomerulus
How is it achieved that all the neurons with one olfactory receptor converge onto one glomerulus?
- Each receptor has a characteristic basal activity. This means when there is no odour around, they still fire at a fixed rate.
- This affects the amount of cAMP in the cell. This is activity independent
- Neurons expressing the same levels of cAMP cause the same level of transcription of a set of guidance cues and their receptors e.g. Robo/slit and Neurotrophin/Sema. These are called type1 molecules
- This means that type1 protein levels are associated with the expression of a particular olfactory receptor
- The axons will grow to the point in the gradient where there is no net repulsion or attraction based on the gradients of type1 molecules present
- This results in sorting of the axons so that axons expressing the same receptor will gather together
Give evidence that expression of a particular receptor decides where the axon leads to?
- They tested this by swapping the receptor that is expressed using knockout techniques. They swapped P2 for P3
- Showed that if this happens, you shift where the axons are navigated to. There is a clear indication that which receptor is being expressed effects where the axons are navigated to
What happens if disrupt type 1 molecules (guidance cues)?
Disruption of guidance cue expression disrupts regional mapping in the olfactory bulb
- Two different olfactory receptor genes with two markers
- If change levels of guidance cues, change where the axons expressing these receptor genes navigate to
How are olfactory neurons organised from the glomerulus?
- Axons entering the olfactory bulb are pre-sorted due to type1 cues. This is activity independent
- Activity then drives higher cAMP levels which turn on the expression of type II cues: Homophillic adhesion molecules (Kirrels and contactins) or mutual repellents (Ephs and Ephrins)
- These interactions sort axons expressing the same olfactory receptors into groups from the glomeruli
Give an overview of how olfactory neurones are guided to the olfactory bulb?
Each olfactory epithelium cell is expressing one type of olfactory receptor and neurons expressing the same receptors go to the same glomerulus. This is activity independent and is derived from olfactory receptor specific expression of typwe1 molecules. Then becomes activity independent which controls expression of type. II molecules
How does the organisation of the olfactory system differ from the visual system?
Unlike the visual system, mitral cell axons projecting to the piriform cortex (PC) do not exhibit any spatial organisation
Give evidence for axons projecting to the piriform cortex not exhibiting spatial organisation
Miyamachi et al, 2011)
- They used transynaptic tracing, injected viruses which can jump across synapses and allowed tracing of neurons
- Axons from the glomerulus were going everywhere with no apparent organisation. Individual odorants are activating subpopulations of neurons in the piriform complex
How does the piriform complex respond to odourants?
- Different odorants don’t just activate one type of receptor
- In the olfactory bulb, an odorant can activate multiple receptors but neurons in the piriform complex responds to structurally dissimilar odorants
Are responses to odours learned?
In mammals, most odours only drive behaviour after learning – learnt by association
Few are innate
Give a study that asks if the piriform cortex (PC) the site of olfactory learning?
Choi et al (2011)
What did Choi et al (2011) do?
- Used optogenetic activation of arbitrary subsets of PC neurons
- They introduced channelrhodospin (ChR2) into subset of PC neurones. ChR2 is a light-activated cation channel that stimulates action potentials upon exposure to light. i.e can ‘fire’ PC neurons independent of mitral cell input.
- They stimulated the ChR2+ subset of neurons with light, paired with either an aversive or appetitive (appetite inducing) stimulus in naïve (unconditioned) animals (classic associative learning).
- After this conditioning, they tested whether the light stimulus alone can elicit the appropriate behavioural response
How did Choi et al (2011) inject channelrhodopsin into the piriform complex?
Three ways of injecting channelrhodopsin into the piriform cortex
- Use synapsin promoter to drive expression as channelrhodopsin. Channelrhodopsin is on the same gene as a fluorescent protein. Uses IRES sites to express both proteins. This hits 50% of the cells at the injection site
- Inject floxed Chr2 into a mouse in which Cre is driven from Emx1 promotor (excitatory neuron-restricted). Normally, a floxed gene is knocked out by cutting and re-joining the DNA strand leading to excising of the gene. However, in this technique, the recombination sites have changed orientation (facing inwards) meaning that the gene isn’t knocked out but it is flipped so it becomes under the control of the Emx1 promotor and can drive expression. This hits 50% of the cells but only excitatory neurons
- Inject foxed Chr2 at same time as virus containing synapsin driving cre. This resulted in a much lower Chr2 expression rate
What did Choi et al (2011) find?
- ChR2 activation can condition aversive behaviour. Photo stimulation of ChR2-expressinf neuron in the piriform cortex was paired with a foot shock
- Animals then exhibited flight behaviour to the photo stimulation alone but only when ChR2 was present in the piriform neurons
- Conditioning with odorants and PS together showed that subsequently either the PS or odorants could elicit flight
How can piriform cortex stimulation be used and what does this tell us?
- Can use piriform cortex stimulation to train the animals. This tells us the neurons have high plasticity
- The same set of ChR2-expressinf PC neurons can be retrained in either direction. This tells us that the cortex is highly plastic
Does the plasticity of the piriform cortex prove that is the site of odorant learning?
- No, just shows that the piriform cortex can be used for associative learning and that it is very plastic
- This is a specific property of the piriform cortex. Other regions of the cortex are not as plastic
- However, it is strongly suggested that random connections from the olfactory bulb into the piriform cortex are used to associate odours with experiences
Give examples of odours that have innate responses?
- trimethyl-thiazoline (TMT) from fox elicits fear in naive mice
- There are spatially invariant projections from the olfactory bulb to cortical amygdala that may be involved
What did Yokoyama et al, 2011 investigate?
Elimination of Adult-Born Neurons in the Olfactory Bulb Is Promoted during the Postprandial Period