TS6: The Nervous System Flashcards
What differentiation processes does a postmitotic neuron go through? (MADSS)
- Migrating to its final destination
- Axon extension
- Establishing dendrite branching patterns
- Forming synapse connections
- Modifying synapse connection
What is the function of astrocytes in postmitotic neuron differentiation?
Facilitate synapse formation
What is the function of oligodendrocytes in postmitotic neuron differentiation?
Myelinate axons.
What is synaptic pruning and why is it important?
Synaptic pruning is the process of eliminating weak or unnecessary neural connections in the brain. It is a natural and important process that occurs during brain development, particularly during childhood and adolescence.
Synaptic pruning has been linked to a variety of neurological and psychiatric disorders, including autism, schizophrenia, and depression. These conditions are thought to be associated with abnormalities in synaptic pruning, which can lead to the development of dysfunctional neural circuits.
What is an axon growth cone?
The axon growth cone is a highly dynamic structure that contains a complex network of cytoskeletal filaments, including microtubules and actin filaments.
The growth cone is responsible for guiding the developing axon to its target cell or destination during neural development.
What are the 4 methods of axon guidance? Give an example protein for each.
- Long-range chemoattraction
- Long-range chemorepulsion
[ligand is soluble]
e.g., DCC/netrin and Robo/Slit - Contact-mediated chemoattraction
- Contact-mediated chemorepulsion
[ligand is cell-bound]
e.g., Eph/ephrin and Unc5/FLRT
What are the four ‘classical’ axon guidance systems?
Slits (bind Robo receptors)
Netrins (bind DCC and Unc5 receptors)
Ephrins (bind Eph receptors)
Semaphorins (bind Plexins)
How did forward genetics give insights into axon guidance for crossing the midline?
Drosophila mutants were identified that, when stained with an antibody that recognizes all axons, showed interesting phenotypes:
- Slit mutants showed axon collapse at the midline
- Roundabout (Robo) mutants showed neurons crossing the midline multiple times
- Comm mutants showed no axons crossing the midline
Forward genetics was then used to take these mutants and find the proteins responsible for these phenotypes, as well as how they regulate midline crossing.
Describe the functions of Slit, Robo and Comm in midline crossing.
How do these control axonal crossing of the midline?
Slit is a secreted protein produced by midline glia that acts as a repulsive axon guidance ligand.
Robo is a receptor for slit on the axon growth cone.
Comm acts in the secretory pathway to downregulate cell-surface expression of Robo.
- Comm prevents Robo from being on the cell surface, enabling axons to cross the midline.
- After midline crossing, downregulation of Comm results in upregulation of Robo, preventing axons from recrossing the midline because of Slit repulsion.
How do we test whether a protein elicits attractive, adhesive or repulsive responses?
The confrontation assay (time-consuming and difficult to quantify)
Stripe assay
State the procedure involved in a stripe assay.
- The protein under investigation and a neutral protein are immobilized on a culture dish in an alternating stripe pattern.
- Cells are seeded on these stripes.
In control experiments, both stripes contain neutral protein only.
How are cortical layers formed?
- At early embryonic stages, neural progenitors in the ventricular zone (VZ) divide symmetrically, expanding the progenitor pool
- At later stages, they become radial glia.
- Neurons produced from radial glia cells via asymmetric division are the first to initiate and complete migration, settling in the deepest layer.
- Each time a neuron is ‘born’, they attach to the radial glia cell and migrate upwards towards the cortical plate
How is neuronal migration regulated by FLRT? What four experiments have aided in uncovering this?
FLRTs play important roles in regulating the timing of neuronal migration.
- Stripe assays have shown that Unc5 forms a repulsive interaction with FLRT, whilst latrophilin has an adhesive interaction.
- Point mutations were then used with structure-based protein engineering, such that mutant FLRT could no longer bind Unc5. This stops the repulsive signal, showing that FLRT signals through Unc5 receptors.
- Structural biology was also used to show that FLRT and Unc5 form a ternary complex with Latrophilin, due to Unc5 and Latrophilin having different binding sites on FLRT. XRC showed they form a 2:2:4 super-complex.
The ability to form different complexes allows for different functions as the neuron migrates.
- RNA hybridization assays showed that as neurons migrate, there’s different expression levels of the receptors, implying each complex only has functions as certain stages of migration to aid navigation.
What is the FLRT-Unc5-Latrophilin supercomplex, and how was it studied?
What did these studies reveal about the supercomplex?
The interaction of FLRTs with Unc5 and LPHN has been shown to promote the repulsion of axons from regions of the developing nervous system that are rich in these proteins.
Multi-angle light scattering (MALS) and NMS was used to study to determine oligomer states of the complex, showing that not all homologues form this super-complex.
This means that the receptors can form higher order complexes (not just 1:1 interactions) and the same protein can engage in different complexes with different functions.
What can Latrophilin, FLRT, and Unc5 bind to?
Why is having so many interaction partners important?
Latrophilin: FLRT and Teneurin
FLRT: Latrophilin and Unc5
Unc5: FLRT and GPC3
Receptors form different complexes, depending on which binding partners are available. This combinatorial code enables relatively few receptors to elicit many different, nuanced cell responses.
Describe the nanofiber assay for studying latrophilin. What did it show?
Nanofibers were used to mimic radial glia cell fibers and were coated with different proteins to monitor how neurons migrate. Antibodies could be used to visualize this migration.
Together with other experiments, it showed that Latrophilin is repulsive for neurons expressing FLRT and Teneurin.
What is in-utero electroporation?
In-utero electroporation (IUE) is a technique used in developmental neurobiology to manipulate gene expression in the developing nervous system of embryonic animals.
It involves the injection of a plasmid DNA construct containing the gene of interest, along with a fluorescent protein marker, into the developing brain of an embryo via a small incision in the uterine wall. The DNA construct is then delivered into a specific region of the developing brain using a glass micropipette, and a series of brief electrical pulses are applied to the embryo to facilitate the uptake of the DNA construct into the cells.
Once the DNA construct is taken up by the cells, it can be expressed, allowing the researchers to visualize and manipulate the expression of the gene of interest in the developing nervous system.
How are nanobodies being used to study neuronal receptor interactions?
Nanobodies are derived from single-chain camel antibodies, and these are being used to interfere with specific protein binding surfaces, hence weakening receptor interactions.
It’s harder to find those that stabilize these interactions, but not impossible, and so these can be used to enhance the receptor interactions.
How could nanobodies influence cancer cell migration?
Stripe assays show that the repulsive interactions seen between GPC3 and Unc5 are found in many cell types outside of the brain, including in cancer lines.
In vivo models have shown that different nanobodies (nano-glue or nano-break) either inhibit or promote the collective migration of neuroblastoma cancer cells, suggesting the tech could be used in cancer therapy.
Describe the structure of vertebrate retina.
The vertebrate retina is a layered structure made of 5 classes of neurons.
The input layer at the back of the retina consists of photoreceptors that detect photons and convert them to electrical signals.
The output layer comprises retinal ganglion cells, which transmit information from the eye to the brain, making up the optic nerve.
In between are bipolar cells and amacrine cells, whose actions influence the signals transmitted from photoreceptors to bipolar cells and then to RGCs.
Pigment cells at the back of the eye absorb extra photons and prevent light scattering.
Describe the type of vision given by the two types of photoreceptors and where they can be found.
Cones are responsible for high acuity, daylight and colour vision; in primates, cones are concentrated in the fovea, the central part of the retina.
Rods are more numerous, more sensitive to photons, and specialized for night vision.
How did psychophysical studies reveal that human rods can detect single photons?
A person in a dark room received a flash of light with varying numbers of photons. The flashes ‘seen’ were plotted over the average number of photons used, taking into account the probability of photons hitting the retina.
For these psychometric functions, a best fit for n was found to be between 5 and 7 photons. Thus, to reliably perceive a flash of light, an average of 5-7 independent photon absorptions must occur in a retinal field of ~500 rods.
Since the probability of 2 photons being absorbed by the same rod under these conditions is small, each rod must be able to report the absorption of a single photon.
How did electrophysiological studies confirm the single-photon response of rods?
A single rod was sucked into an electrode to form a tight seal. Electric current passing through ion channels in the plasma membrane of the segment within the electrode could be measured in response to a light beam.
By systematically reducing the light intensity, a condition was reached in which most light flashes didn’t produce any response. The occasional responses that did occur has a uniform size, and was fitted to a Poisson distribution, showing that the responses were mostly caused by absorption of single photons and occasionally 2 photons.
These measurements confirmed that photon absorption results in hyperpolarization of rods - that is, current flows out of the rod in response to light. Each photon absorption results in ~1pA of net outward current, equivalent to blocking 10^7 position ions that would otherwise flow into the cell….
How is inward flow of current (depolarization) blocked in rod cells?
When light is absorbed by the photopigment molecule, rhodopsin, in the outer segment of the rod cell, it initiates a series of biochemical events that ultimately lead to the closure of ion channels in the outer segment membrane and the reduction of the intracellular concentration of cyclic guanosine monophosphate (cGMP).
The reduction in cGMP concentration causes the cGMP-PDE complex to become activated, which breaks down cGMP into GMP. As a result, the concentration of cGMP in the cell decreases, leading to the closure of the cGMP-gated ion channels in the plasma membrane and the hyperpolarization of the cell.
Describe the structure and function of rhodopsin.
How does the structure of the subunits relate to its function?
Rhodopsins are the photosensitive molecules in rods.
Each rhodopsin consists of an opsin GPCR protein and a small molecule of retinal that’s covalently attached to a lysine residue in the opsin.
Retinal is the chromophore that exists in two isomers. Photon absorption causes a switch of 11-cis retinal to all-trans retinal, triggering a conformational change in the opsin.
This change opens up a binding site for a heterotrimeric G protein, transducin.
Describe the transduction cascade involved in photon absorption within rod cells. How is the signal amplified?
- Light triggers the isomerization of retinal that causes a conformational change in opsin.
- Transducin is then able to bind opsin and become activated.
- Activated transducin catalyzes the exchange of GDP for GTP, releasing the alpha subunit.
- Alpha-GTP activates phosphodiesterase by sequestering the inhibitory subunits.
- PDE hydrolyzes cGMP to GMP.
- Low cGMP levels result in CNG channel closure, hyperpolarization of the cell, and a decline in glutamate release.
- System reset.
Amplification occurs through 1 rhodopsin activating >20 transducin molecules, each of which can activate PDE.
How do cGMP/GMP concentrations control membrane potential? How was this discovered?
The reduction in cGMP concentration causes closure of the cGMP-gated ion channels in the plasma membrane and the hyperpolarization of the cell.
Discovered using patch-clamp to change the levels of cGMP and monitor channel conductance, as well as cloning of the gene encoding the gated channel (CNG channel).
Describe the structure and function of CNG channels.
Its primary structure resembles that of K+ channels, with 6 TM domains and a pore loop between S5 and S6.
Like K+ channels, functional CNG channels comprise 4 subunits with 4 cGMP binding sites.
Its opening is non-selective (like nicotinic ACh receptors), but due to its reversal potential, Na+ influx exceeds K+ efflux. Hence, the net effect is depolarization when cGMP is bound.
Describe the process of recovery in rod cells after the phototransduction cascade.
- The closure of CNG channels reduces [calcium], which causes GCAP to activate guanylate cyclase, leading to increased cGMP production.
- cGMP binding to CNG channels leads to channel opening and membrane depolarization. FEEDBACK LOOP.
- RGS9 (GAP protein for transducin) facilitates hydrolysis of alpha-GTP, thereby deactivating PDE.
- Rhodopsin kinase specifically phosphorylates rhodopsin. Phosphorylated rhodopsin recruits binding of arrestin, deactivating rhodopsin.
What is Weber’s law? How does this relate to light adaptation of rods?
The ‘just-noticeable’ difference between two stimuli is proportional to the magnitude of the stimulus.
In the visual system, this means that photoreceptors become less sensitive to the same intensity of stimulation when the background illumination is higher.
In other words, to achieve the same amount of hyperpolarization at higher background illumination, a stronger stimulus is required.
How does the light adaptation of rods work at the molecular level?
High levels of background illumination cause some cGMP-gated channels to close, resulting in a decline in [calcium]i. This decline leads to the following biochemical changes:
- The basal level of GC activity increases because of the action of GCAP (cGMP increases).
- Activated rhodopsin phosphorylation increases because it’s normally inhibited by high [calcium]I, which leads to more arrestin binding that competes with R* binding with transducin-alpha.
These changes make phototransduction less efficient, and thus stronger activation of PDE by more light is required to promote effective hyperpolarization.
NB: these events occur in the outer segment of the photoreceptor, so [calcium]i changes don’t complicate its role in regulating synaptic transmission, which occurs in axon terminals at the opposite end of the photoreceptor.
How does high-acuity and color vision work?
Cones are highly concentrated in the fovea at the center of the primate retina, hence why color can be seen best at the center of the visual field and not well at the peripheries.
How do we know that cones are less sensitive, but much faster than rods?
Comparing responses to light flashes in single rods or cones revealed cones are much less sensitive than rods.
Cones also recover faster than rods and produce an ‘undershoot’ recovery phase.
What are the 3 types of cone cells in humans, and why are they needed?
How do they detect different wavelengths?
S-cone
M-cone
L-cone
The presence of all 3 cones is necessary for humans to perceive a full range of colors and to have high-acuity vision.
Different cones express different opsins, allowing them to recognize different wavelengths.
