Term
| What is the evidence that neurotransmission is not essential in axonal navigation? |
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Definition
| Deletion of Munc18-1, which is required for neurotransmitter release from synaptic vesicles in development, does not prevent normal brain organization. |
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Term
| What are the criteria for a protein to be considered responsible for axonal guidance? |
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Definition
1) Ligand and receptor expression in particular axon pathway at the time of pathway development (place and time)
2) Supports or inhibits growth in vitro
3) Blocking experiments |
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Term
| What factors determine the excitability of a cell? |
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Definition
Requires "resting potential" and ability to create depolarization that leads to an AP.
Number, type and distribution of voltage-gated ion channels in membranes determines voltage fluctuations in response to incoming stimuli |
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Term
| How are neuronal resting potentials established? |
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Definition
1) Extracellular potassium becomes regulated by glial pumps that reduce it from 35 mM to 3 mM (K is high inside cells).
2) Development of K+ channels that are open at rest. |
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Term
| What ions are responsible for first APs in neurons? |
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Definition
Calcium channels!
After K+ channels prime membranes for excursions in positive direction, Calcium channels allow membrane depolarizations.
In from neurons, this happens 8 hours after last round of DNA synthesis! |
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Term
| What underlies the switch from slow to fast AP development? |
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Definition
| Switch from use of slowly inactivating Ca2+ channels to Na+ channels and delayed rectifier K+ channels (100 ms to 2 ms!) |
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Term
| What is the evidence in support of the switch from slow to fast AP generation being intrinsically programmed? |
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Definition
Rohon-Beard cells in frog embryos of the neural plate (large sensory neurons) switch expression of slow Ca++ channels to Na+ channels and drK+ channels even when they are dissociated.
RNA and protein synthesis are required, as is contact between neural and mesodermal cells (that will develop the notochord). |
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Term
| How can the "cleavage-arrested blastomere" model be used to address the requirements for the development of fast excitability in neurons? |
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Definition
Embryos are arrested at 2-32 cell stage , causing them to give rise to cells that express neural, muscle or epidermal proteins, instead of hundreds of progeny of a given fate.
- Cell cleavage is inhibited with Cytoclasin-B and sister blastomeres are aloud to develop.
- This model enabled the discovery that contact between neural and mesodermal cells is required to develop Na+ spikes and large K+ currents (delayed rectifier) from slower Ca spikes. |
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Term
| What is the role of proteases in the transition from slow to fast AP generation? |
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Definition
Proteases are needed to modify integrin molecules so that neural-mesodermal interactions can occur.
1) Subtilisin-like endoproteases (convertases) are required to induce post-translational cleavage of extracellular domains from integrin alpha subunits that are required to establish adhesion and signal transduction.
2) These proteases are secreted from mesodermal cellos to induce nerons to develop ion channels at later stages. |
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Term
| What is the evidence for developmental roles of slow Ca++ action potentials? |
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Definition
- If you inject K+ channel mRNA into 2-32 cell embryos (forcing their expression earlier than usual) and look 8 (normally slow AP) and 24h (normally short AP) later, you see that short AP exist at both time points.
- These neurons have fewer neurites and don't appear to differentiate as well in vitro, suggesting that the TIMING of fast APs is critical |
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Term
| What is the functional role of calcium APs at the time in development when there are no synapses? |
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Definition
Growing neurites require calcium to establish cytoskeletal scaffolds and membrane in growth cones.
- Growth cones will stop growing if you induce trains of APs (calcium waves) in large sensory neurons from frog embryos
(set point)
- Calcium activates phosphatases (calcineurin), which dephosphorylate GAP-43 is growth bones, preventing the stabilization of actin filament polymerization. |
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Term
| What is the role of the Kca channel that emerges later in development during synapse formation? How do they differ from Kdr? |
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Definition
Experiments performed in chick parasymapthetic (CG) neurons
1) Kdr channels create fast activating currents that keep APs short
2) Kca channels create long AHPs and space bursts.
- Unlike Kdr, Ca and N channels, Kca channels do not develop is dissociated cultures, indicating that they require external environmental signals.
- In vivo, pre-ganglionic cells release beta-neuregulins, which are growth factors that bind to receptors on CG neurons. These factors are necessary, but P-synthesis is not.
- Factors released from target are also critical for Kca development.
**these factors may induce post-translational modifications of existing K+ channel subunits to change their calcium sensitivity** |
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Term
| True or False: Synapses do not form with inappropriate targets. |
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Definition
True! The pathfinding process is accurate.
More synapses form than are needed, however, and many are pruned later. |
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Term
| What about chemical communication is unique to synapses in neurons? |
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Definition
1) Fast (ms speed)
2) Focal (only small part of single cell) |
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Term
| What is the evidence supporting the development of pre- and postsynaptic function prior to synapse formation? |
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Definition
Experiments in Xenopus spinal nerve and muscle co-cultures.
1) Muscle cells stained with fluorescent BTX show clusters of post-synaptic receptors
2) "Sniffer" pipettes detect presynaptic neurotransmitter release (outside out patch of AChRs from muscle cells brought close to pre-synaptic neurons to detect ACh release) |
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Term
| What are the major events that occur during synapse recognition? |
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Definition
1) Muscle makes and secretes growth factors (bFGF), which act via TK signaling to attract nerve fibers to form synapses (up-regulate synaptotagmin)
2) Simple contact with targets via CAMs (cadherins) causes nerves to dramatically increase NT release
3) Increased NT release excites muscle cell and increases Ca levels, causing muscle cell to release growth factors (BDNF)
- BDNF binds Trk receptors on cholinergic neurons, speeding up maturation.
**Without BNDF, formation occurs without maturation** |
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Term
| What is the role of BDNF is synapse maturation? |
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Definition
In xenopus NMJ system,
1) Synapses form and neuron adheres to muscle with CAMs
2) Adhesion causes increased ACh release, which causes calcium to accumulate inside muscle cell
3) Calcium in muscle cell leads to secretion of BDNF, which binds Trk receptors on neurons.
4) BDNF-Trk interactions are critical for maturation (block it, and you lose it). |
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Term
| What post-synaptic specialization occurs following NM innervation? |
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Definition
- Upon contact, existing ACh receptors become concentrated at synapse, independent of ACh binding to receptors!
- Nerve secretes AGRIN, which binds synaptic basal lamina and causes AChR aggregation.
- AGRIN-mediated receptor re-distribution requires MuSK (muscle specific TK) |
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Term
| How are multiple axons innervating the same nerve terminal area on one muscle fiber reduced to a single axon? |
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Definition
Activity-dependent synapse elimination (Not death)
Aysnchonrous activity between inputs increases the speed of elimination
- in NMJ culture, two synapses on a single muscle fiber that are stimulated asynchronously leads to heterosynaptic competition.
- Activated muscles release substances that eliminate inactive synapses, but not active synapses, which are protected by other signaling molecules
Hypothesis 1: Muscle activation at AChR leads to calcium entry and NO synthesis, which can diffuse back to pre-synaptic neurons and act on active and inactive neurons differently (blocking NO blocks heterosynaptic competition)
Hypothesis 2: Muscle activation of AChR and calcium entry leads to post-synaptic release of proBDNF. If processed to BDNF, it will bind TrkB receptors and cause synaptic maturation. If remains as proBDNF, will bind p75 receptor and cause tretraction. Key is that active neurons secrete MMPs that can process pro-BDNF! |
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Term
| Describe the functional organization of the biosynthesic, endocytic and retrieval pathways of the ER. |
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Definition
1) Biosynthetic: proteins translocated from ER to PM (COPII pathway) or to lysosmes via late endosomes.
2) Endocytic: Molecules ingested in PM-derived vesicles delivered to early endosomes and then to lysosomes (via late endosomes).
3) Retrieval pathway: Late endosome to Golgi and then to ER (COP1 pathway). From early endosomes to PM. |
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Term
| How is the GTPase Sar1 involved in the biosynthetic pathway of the ER? |
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Definition
COP-II mediated transfer of proteins from ER to PM requires Sar-1 and is required for normal rates of axonal outgrowth
- GTPases are activated/inactivated by GEF (switch of GDP for GTP) and GAP proteins (GTP hydrolysis), respectively.
1) Cytosolic Sar1 is activated by Sec12 (GEF), which is embedded in ER.
2) GTP-bound Sar1 exposes fatty acid tail that inserts into ER and anchors it.
3) ER-bound Sar1 recruits COP-II coat proteins to ER and leads to budding of vesicles.
4) GTPase activity (initiated by GAPs) is required for uncoating, which is required for fusion. |
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Term
| How is Sar-1 involved in axonal outgrowth (what is the evidence?) |
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Definition
Sar-1 is GTPase required for COP-II-mediated biosynthetic ER pathway.
- Sar1 is targeted and concentrated in growing axons in culture.
- Over-expression of WT-Sar1 (Sar1-Flag fusion protein) in newly plated neurons increases axonal growth rates
- Over-expressed Sar1 mutations in GTP binding site in newly plated neurons inhibits axonal outgrowth
- Sar1 shRNAs (block translation of mRNA) disrupts axonal elongation (Golgi is disrupted but retrieval pathway remains normal).
- COPII coat components concentrate in axon during periods of localized axonal growth. |
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Term
| Describe the neurotrophic factor hypothesis |
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Definition
NTF hypothesis
1) Target-derived secreted factors act at a distance
2) The amount secreted is proportional to the size of tissue
3) Competition for survival factors matches the number of neurons to the size of the target. |
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Term
| What are the tropic and trophic effects of NGF? |
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Definition
1) Tropic (local)
- Increases axon growth and acts as an attracting guidance cue
- Alters cytoskeletal dynamics
2) Trophic (transport to soma)
- improves neuronal survival and transcription |
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Term
| What is the functional role of the p75 neutrophin receptor? |
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Definition
Low-affinity receptor that binds all neutrophins (NGF, GDNF, NT3 and NT4).
Growth or Death depending on context
- Enhances NGF activation of trkA
- In absence of trK receptors, p75 promotes apoptosis |
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Term
| What are the members of the GDNF family and what is their role in neural development? |
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Definition
Like Neurotrophins, GDNF, Neurturin and Artemin are expressed by sensory neurons during target innervation.
They bind GFR receptors with different alpha subunits, and activate adjacent Ret TKs.
1) GDNF supports axon growth and survival of dopaminergic neurons in substantia nigra. |
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Term
Functionally diverse sensory neurons innervate different peripheral tissues and convey different types of information to the DRG.
What types of factors are responsible for this diversity? |
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Definition
Expression patterns of trophic factors.
1) Neurotrophin family (NGF, BDNF, NT3, NT4) binds trk receptors
2) GDNF family (GDNF, neuturin and artemis) binds GFRa receptors |
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Term
| How does NGF regulate axon guidance? |
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Definition
1) NGF binds trkA receptor, leading to expression of semaphorin receptor.
2) Semaphorins are secreted proteins that act as short-range inhibitory signals in axon growth cones. |
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Term
| What is the functional importance of the Neurotrophin NT3 vs. NGF? |
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Definition
TrkC/NT interactions are required for survival and targeting of proprioceptive neurons (both cutaneous AND central).
TrkA/NGF interactions are required for Nociceptor development (cutaneous innervation, but NOT central)
- Congenital TrkA deletiion leads to insensitivity to pain |
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Term
| What is the relative role of NGF and GDNF in nociceptor neurons? |
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Definition
Both classes die in NGF/TrkA knockouts!
1) NGF binds TrkA and is responsible for cutaneous innervation and cell survival (not central)
2) Shortly after birth, 50% of nociceptors switch from TrkA to Ret expression (utilizing GDNF)
- Ret is required for cutaneous innervation after birth, but NOT survival |
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Term
| How does NGF get to the nucleus to exert trophic effects? |
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Definition
Theories
1) Retrograde transport of NGF-pTrkA complexes in signaling endosomes (unlikely)
2) Retrograde transport of phosphorylated TrkA unbound to NGF (unlikely)
3) Tetrograde transport of signalling molecules downstream of phosophorylated TrkA. |
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Term
| What are the key differences in trophic factor function for PNS between embryonic and postnatal life? |
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Definition
In the embryo, trophic factors function by establishing functional connectivity and maintaining neuronal survival (NGF-TrkA in nociceptors)
In postnatal animals, neurotrophins refine functional identity of neurons and maintain their functional properties via restricted expression of neurotrophin receptors in functionally distinct populations and sensory neurons. |
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Term
| What observations in chick embryos led to the identification of NGF as a neurotrophic factor? |
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Definition
1) Secreted by target (removal of target tissue/limb bud alters neurons survival)
2) Can act at a distance (survival modulated by diffusible factor from DRG to sensory ganglia)
3) Purification of growth-promoting factor from DRG led to NGF identification, which was shown to be critical for axonal outgrowth. |
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Term
| What are some useful model systems that can be used to investigate axonal navigation? |
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Definition
1) Tracing axonal pathways in vivo during development
2) Transplant of origin or target tissues
3) Co-culture of tissue explants
4) Mutagenesis screens: C. elegans and Drosophila
5) Ectopic expression or knock-out genes |
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Term
| What major groups of proteins are involved in axon navigation? |
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Definition
1) Permissive and non-permissive ECM components (laminin, fibronectin, collagen)
2) Membrane-bound CAMs (N-CAM, Cadherins)
3) Secreted cheotactic and chemorepulsive signals
4) Contact-mediated guidance proteins (GPI ephrins) |
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Term
| What is the functional role of Netrin proteins? |
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Definition
Unc 6, Unc 40 and Unc 5 are netrins that bind to DCC and Unc 5 receptors
Neurons that express netrin receptors are guided to or away from targets by netrin-1.
When you knock out netrin in SC or retina, axons do not innervate their targets.
In response to netrin-1, DCC receptors are attractant and UNC-5 receptors are repulsive |
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Term
| What is the role of Slit and Robo in axonal pathfinding? |
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Definition
Slit is a secreted protein that acts as a repulsive axon guidance cue.
Robo is the transmembrane receptor that binds Slit at leucine right repeats (LRRs)
Flys without Slit-Robo signaling are commissure-less. |
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Term
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Definition
| Repulsive axon guidance cues that activate complexes of cell surface receptors called Plexin and Neuropilins (vs. Slit, which just binds to Robo). |
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Term
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Definition
GPI-linked or transmembrane ligands that bind RTKs on cell membranes of other cells.
Can be attractive or repulsive |
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Term
| Describe how the projection of retinal ganglion cells to the optic tectum of frogs is topographic. |
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Definition
Visual map in tectum is inverted relative to the retina, because axons from dorsal and ventral retina project to ventral and dorsal tectum, respectively.
Axons from nasal and temporal retina project to the posterior and anterior tectum.
When the optic nerve is cut and the frogs eyes are rotated, everything that normally enters the retina dorsally, does so ventrally. Therefore, the topographical organization is uncaged, but the location of the input itself is reversed. |
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