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ZOOLOGY 523 Final
Neurobiology UW Madison Final
Terms in this set (55)
Specializations that occur in the presynaptic and post synaptic cells in the NMJ
- Nerve terminal
- Synaptic vessels
- Active zone
- Synaptic cleft has basal lamina (sheet of extracellular matrix) with Acetylcholine esterase (AChE) breaks down Ach
o The synapse recognizes the pulse of ACh and responds
- Junctional folds
- ACh receptors (AChR)
o AChR's increase the surface area on muscle fibers for more receptors
Life Cycle of ACh
1. Cycle begins with synthesis of Cholien and a choline transporter in the pre-synaptic cell of the neuron; sodium enters the cell here too
2. Choline CoA + acetyltransferase catalyzes to form ACh
3. ACh is packaged and stores in presynaptic vesicles and moves down
4. Voltage sensitive Na channels open, elevate concentration of Na in the cell, cause vesicles to move down cell and fuse with postsynaptic membrane
5. ACh is released into the synaptic cleft
6. ACh binds to nAChR (nicotinic ligand gated receptor) or a muscarinic and causes sodium channel to open and let sodium into post-synaptic cell
7. This causes a rapid biological response in the cell, but is rapidly terminated due to AChE cleaves ACh, creating choline and acetic acid. The process starts over again
development of muscle fiber
- number of myoblasts fuse to form syncytial myotube, each containing several nuclei (=syncytial). each myotube becomes a muscle fiber
- no synaptic specializations on myotube, has low levels of widespread AChR's
- as verce approaches, get connetion of AChR's under axon end, then get special basal lamina
Development of NMJ
- motoneuron axon terminal approaching myotube, site of contact becomes synapse
- also see presynaptic specializations appear, active release zone ACh, etc.
How to follow the development of ACh receptor clusters:
1. Label the "old" AChR's already in the membrane with the alpha-bungarotoxin (alpha-Btx), BINDS IRREVERSIBLY
2. Stain later with antibody so you can see both and newly synthesized AChRs
(location of AChR clusters depends on the nerve)
development of NMJ continued
- clustering due to both movement of old AChRs and synthesis of new AChRs
- mRNA fro new AChRs transcribed in nuclei nearest synapse
- motoneuron cues for AChR clustering and muscle cues for axon terminal are left in basal lamina (extracellular matrix) at NMJU as shown by in vivo experiment on regulation of mature NMJ
experiments for NMJ AChR clustering and muscle cues
1. Cut motoneuron axon, follow site of old NMJ by finding location of old AChEsterase in basal lamina- use here as "marker". Axon grows back to old NMJ
2. Kill muscle fiber, new one regenerates, makes synapse at old site where motoneuron is
3. Cut axon, keep re-cutting. kill muscle. ONly thing left is basal lamina "ghost"
tests of caulality
1. Sufficient (addition): if you add it, will it cause the effect?
2. Necessary (subtraction): what happens iif you remove it? will the effect go away?
3. Selectively removing agrin from muscle or neuron (shows that agrin made by neuron is necessary but that nerve and muscle both make different forms of agrin, so you can take agrin away from only the neuron)
What happens when you reduce clustering by removing Agrin and increase AChRs by removing activity?
can improve loss of AChR clustering caused lack of Agrin by also removing presynaptic ACh (mutate choline acetyl-transferase [chat], required for ACh synthesis)
What is ARIA
- AChR Inducing Activity - it is really just a protein signal call neureguliin
- ARIA sufficient to induce AChR synthesis, but NOT necessary, knockouts normal
What is the receptor for Agrin
- Agrin Receptor = Muscle Specific receptor kinase (MuSK) I (with LPR4)
- Agrin and MuSK necessary for noaml AChR clustering in vivo (use mutant "knockout" mice lacking genes)
Selectively removing agrin from muscle or neuron
1. rat motoneuroncan clustern AChRs in chick muscle, and vice versa
2. have antibodies that bind chick agrin but not rat agrin
what happens when you remove neural chick agrin with antibodies
chick motoneuron can no longer signal to rat muscle
what happens when you remove chick muscle Agrin
rat motoneuron still signals to chick muscle
In vitro bioassay
- used to find AChR
- use extract from electric organ of Torpedo ray because lots of cholinergic synapses
- extract induces AChR clustering in myotubes in vitro
- isolate specific proteins, re-measure for their effects in bioassay, purify further, etc. etc.
protein signaling molecule, made by nerve (and muscle fiber), accumulates in basal lamina
- aggregates ACh
- sticks to collagen
- sits in extracellular matrix and binds to receptors
different signals at other types of synapses
- often complex and overlapping functions for signals. Some diffusible cues, some by binding between transmembrane proteins on pre and post-synaptic cells (Neurexin pre and Neuroligan post) = Cell Adhesion Molecules (CAMs)
- widened region at end of many growing axons
- reads environmental cues transduces that into local stability of cytoskeleton and thus direction of growth
as we age, old connections are deleted through a process called synaptic pruning
"use it or lose it"
- a neural stain that completely darkens a few of the neurons in each slice of tissue, thereby revealing their silhouettes
- fills all processes of small % of neurons
- selective for only some neurons; can follow with a microscope
types of cues growth cones can read
1. physical cues - tubes, grooves which axons grow along (not diffusable)
2. molecular substrate cues (not diffusable) - bound to something along route
a. extracellular matrix (ECM) around cells (not diffusable) - collagen, laminins, fibronectin, etc... axons love basal lamina
b. cues on cell surfaces cell adhesion molecules (CAMS) (not diffusable) - proteins (usually transmembrane) that bind cell to another
3. Diffusible Cues in Substrate: not stuck, but free to diffuse through the medium that the nerve cell is growing in and provide some attraction
4. repellant cues -
- addition of new material mostly (but not entirely) at tip of axon
- Two methods of testing:
1. direction of outgrowth controlled by local stability of cytoskeleton
2. adhesion of pioneer axons to guidepost neurons
- later growing axons can stick to (fasciculate with) specific early-growing PIONEER axons
- specificity controlled by expressiion of specific CAMs on specific axons
- immature nerve cells, cell bodies act as a guidepost
- pioneer neuron is going to decide where to grow because it's going to see guidepost and adhere to it
Axon Guidance and Target Selection
example: retinotopiic map in optic tectum - temporal (posterior) retina to anterior (rostral) tectum
axes in body
rostral = nose
caudal = tail
dorsal = back
ventral = belly
dorsal-ventral direction in head
superior/inferior and anterior/posterior
- superior = up, inferior = down, straight line in human
- anterior = front, posterior = rear. In fish it's same as rostral-caudal, but not in humans
- give up top of human head is "superior" or "dorsal", front of head is rostral
nasal = near nose, is anterior in fish
temporal = near temple, is posterior in fiish
- retinotopic order preserved during regeneration of half retina axons onto tectum - due to "chemospecificity"
1. remove half of retina, cut optic nerve, allow remaining axons to regenerate to tectum
2. different nerve cells have a different affinity for differnt chemicals
the "black pit" of developmental neurobiology
- 1970s: many experiments trying to figure out what and where cues were? cues vs the timing of outgrowth? cues on retinal axons or on tectum or both? stable cues, or did they change after surgeries?
- often contradictory results, fish vs frog, regeneration vs original outgrowth, surgery to retina or tectum or both, lab vs. lab
what is the stripe assay and what type
- grow retinal axons on cell membranes derived from different parts of tectum
- temporal axons prefer anterior tectal membranes but not if proteins in posterior membranes are destroyed
- so temporal axons are inhibited by contracting substrate cue in posterior tectal membranes
What are some possible reasons why the nasal axons grow into the posterior instead of the anterior of the tectum?
- Nasal axons have very low amounts of EphA receptors, so they are pretty much insensitive to the repulsive cue from the EphrinA
- So, by defaut, they grow to the posterior
- Temporal axons have high concentrations of EphA receptors, so they are repulsed by the posterior and grow towards the anterior
What is meant by reverse Ephrin-Eph signaling? Give an example.
- Forward signaling: Ephrin is the signal, Eph is the receptor
- Reverse signaling: Eph is the signal, Ephrin is the receptor
- Nasal axons with high EphrinA might be repulsed by high EphA on temporal axons or anterior retina
Give an example where a member of Ephrin protein family is used as an attractive guidance cue
Dorsal-ventral cues: Ephrin-B and Eph-B attractive, uses both forward and reverse signaling
animal development: first steps
- Fertilized egg divides, cells move to form embryo
- First divisions = CLEAVAGE, cells do not move, can occur without growth
- Produces a BLASTULA
Germ Layers in gastrula (second steps of animal development)
- Ectoderm- outermost, makes skin, nervous system
- Mesoderm- middle, makes muscles, internal organs
- Endoderm- innermost, makes digestive system
gastrulation (step 3 animal development)
forms germ layer via cell movements
- In frogs, mesoderm invaginates (folds inward to make cavity) around the blastopore
- Dorsal mesoderm derived from "dorsal lip of blastopore" and becomes "roof" of archenteron (cavity)
- Nearby "neurogenic" dorsal ectoderm forms CNS
organogenesis (step 4 animal development)
germ layers subdivide to make organs
- Ectoderm splits into skin (epidermis), neural tube and neural crest
- The neural tube forms the CNS- brain and spinal cord
Forming the neural tube
- Dorsal ectoderm thickens into NEURAL PLATE, invaginates (folds inward) to form long NEURAL TUBE, makes CNS (brain, spinal cord)
- Occurs dorsal to dorsal mesoderm, part of which makes NOTOCORD- mesodermal "rod"
o Chordate maker, mesodermal rod, plays a skeletal role, makes big cells with big vacuoles filled with water
- Cells at interface between tube and dorsal epidermis (skin) make NEURAL CREST- migrate to make peripheral neurons and others
the mechanisms by which cells become different from each other:
Different changes happen in different locations to different cells:
- In many embryos, can relate the early position of a cell to the eventual Fate of the cell (what the cell or ots progeny will DIFFERENTIATE into)
- So, how do different cells get SPECIFIED for a different fate
o Inherited information vs. signaling between cells
What is a cytoplasmic determinant, and how does it specify inherited, intrinsic identity of cells?
Inherited information- all cells have the same DNA, so it must be something else:
Cytoplasmic determinant: molecule (mRNA, protein, etc.) inherited from parent cell by only one daughter cell, makes that cell different
- Cytoplasmic determinants are often transcription factor proteins, or the mRNAs that encode transcription factors
o One cell inherits transcription factor, regulates mRNA synthesis of target genes
o Makes or fails to make proteins, which changes cell identity
How is signaling used to specify cell identities? What is a diffusible morphogen?
Test: take cells away from their normal neighbors (isolate them, place them in abnormal ("ectotopic") position in embryo, remove neighbors, etc.)
- Do the cells make the same tissues they normally make, or different? Do they change what their neighbors make?
o Same- probably relying on inherited information- cells have been "determined"
o Different- responding to signaling between cells
- Morphogen = a substance whose non-uniform distribution govern the pattern of tissue development in the process of morphogenesis or pattern formation
How can you use transplantation of a cell to an abnormal position to decide if it's fate depends on signaling?
Signals from one set of cells changes adjacent cells
- Dorsal mesoderm signals to any nearby ectoderm to make neural tube (even after transplant)
2. Subsequent development
a. Forms secondary (induced) embryo
3. Primary structures formed opposite of each other
What cells give rise to the neural crest? Where are they found?
Cells at boundary between neural plate and epidermis become NEURAL CREST
- Neural crest cells migrate away from neural tube, make most neurons and glia found outside the CNS
- Almost all PNS cells come from outside the CNS
o Ganglia: dorsal root, sympathetic parasympathetic
o Enteric (gut) nervous system
o Glia of nerves (Schwann cells) and ganglia
o Non-neuronal tissues as well, endocrine, mesoderm in head
What signals help generate the neural crest cells within the ectoderm?
Neural crest specified by BMP, Wnt, and FGF signaling between the neural plate and epidermis cells
What is meant by epithelial-mesenchymal transition? If you don't know what an epithelium or a mesenchyme is, you might want to look it up.
Direct migration of crest cells
- Cells undergo "epithelial to mesenchymal" transition (EMT)
- Cues often same as used by axon guidance. Migrate away from high Ephrin B, Semaphorin
Is neural crest migration random, or does it follow cues?
- Follow cues: the cues are often the same used by axon guidance
- Migrate away from high Ephrin B, Semphorin
How is a chick-quail chimera used to trace the normal fate of neural crest cells? How is it used to test whether pre-migratory neural crest cells are already fixed in their eventual fate?
Early Research: use chick-quail chimera to follow neural crest fates
- Transplant piece of chick neural plate/tube into quail or vice versa
- Can tell the difference between host and donor piece by nucleoli or antibody
- Or make neural tube and crest. Ells fluorescent
What is the evidence that pre-migratory crest cells can switch between sympathetic and parasympathetic fates? What was originally used to indicate which fate the cells chose?
Do crest cells have fixed fates before migration? Analyze using transplants
- Can switch sympathetic (adrenergic) vs. parasympathetic (cholinergic) fates by transplanting to new location or supplying signals
- Originally used in chick quill chimera
Are the fates of all neural crest cells unfixed, or are some fixed before migration?
But... some crest fates are set by anterior-posterior position prior to migration
- Especially true for cranial (head) neural crest, has some unique non-ectodermal fates: cartilage and muscles of jaw, etc.
- Crest and neural tube express specific transcription factors along anterior-posterior axis esp. HOX genes, provide spatial memory
Last axis - Radial (inside to outside)
- Making layers in brain
- Most cell division limited to inner (ventricular and sub-ventricular) zones in neural tube
- Some cells stop dividing migrate our of inner zone to outer-most zone. Others stay in division zone and make mor precursors
What are the two precursor cell types in the ventricular zone? What is a stem cell?
1. Dividing radial "glia"
a. Produce immature neurons and dividing intermediate precursor cells (IPCs). Radial glia are self renewing
2. IPCs (Intermediate precursor cells)
a. also are self-renewing, but some progeny stop dividing and migrate outward along radial glia, are immature neurons and glia
what does it mean to be Self- Renewing
Self- Renewing: Radial glia and Intermediate Precursor Cells can divide to make 2 dividing cells, or 2 non-dividing cells, or a dividing and a non-dividing cell
- = STEM CELL - cell that can produce both differentiated and dividing cells
- Asymmetric cell division can produce two different fates
What labeling technique is used to birthdate post-mitotic cells in the developing neural tube?
Birth-dating neurons by:
- Inject pulse of H3-thymidine or BRDU to label DNA in S phase
- Diluted in cells that keep dividing, strong in cells that stop dividing
- Look at mature brain, see where radioactive cells are
- Innermost layer born (post-mitotic) first, outer layers formed last
*** trying to figure out the stage at which a precursor for a particular nerve cell becomes post-mitotic (no longer able to divide)
In what layers of the brain do post-mitotic cells wind up if they are born early in development? What if they are born later in development?
- Cells migrate first to inner layer
- Later-born cells migrate to more outer layers
- Later-born neurons migrate from ventricular zone (layer 6) past early-born neurons to outermost layers
How are heterochronic transplantation experiments used to test where and when cells acquire layer-specific identities? Do cells from a young ventricular zone behave the same as cells from an older ventricular zone in these transplantation experiments? Are cells acquiring layer-specific identities before or after migration into a layer?
What is a heterochronic transplantation experiment?
- Transplant VZ cells between embryos of different ages (heterochronic transplants)
- Try to transplant a cell from a young animal into an older animal and vice versa
- Take cells form an early stage (normally makes inner most layers) and transplant it into late cell (aka an older animal) that already has those innermost layers and is now in the process of making outermost layers
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