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DEVB3001
| Question | Answer |
|---|---|
| What were the two lines of evidence that indicated that zika virus was responsible for microcephaly | 1. Microcephaly cases in Brazil increased around the time of zika transmission 2. Researchers found traces of the virus in the amniotic fluid, brains and spinal fluid of babies with the disease |
| What type of virus is Zika? | Single stranded sense RNA in a protein shell (capsin) and surrounded by a lipid-protein envelope |
| Describe the progression of stem cells in the brain | Radial glial cells (stem cels of the neuro-epithelium_ slowly differentiate into transit amplifying cells, which quickly differentiate into post mitotic igratotry neurons, which differentiate into outer radial gilal clles |
| What is lisencephaly? | Failure to develop the gyres of the brain |
| When does neurogenesis occur | between 2-4 months |
| When does the the majority of dendritogensis, myelinatin and synaptogenesis occur | after 7 months and post-natally |
| When does axonogenesis occur | between 2-7 months |
| what are the three postulates that researchers need to fulfill to prove that zika virus causes microcephaly | association between zika virus and microcephaly isolation of virus animal models which show that the virus is pathogenic |
| Was there microcephaly in micronesia? | No only in brazil, indicating that some mutation must have occured |
| Why does zika virus target the brain | It's receptors are there |
| How did zika affect mice? Why are mice bad models for zika induced microcephaly? | It reduced cortical thickness but not microcephaly. They have a short gestation time. |
| Describe the zika experiment with the induced pluripotent stem cells. What did they conclude | IPSC converted to neuroprogenitor cells and organoid. Infected organoid resulted in cell death. Infected NPCs died and failed to differentiate. Neurons did not die. Therefore viral portein inhibits replication and differentiation of NPC |
| How do you make induced pluripotent stem cells | Take fibroblasts and add transcription factors |
| How did zika virus affect aborted babies (aborted tissues) | the virus preferentially infected radial glial cells |
| What is special about mitocohondria?Do they create their own nucleosides? | They have their own DNA. No they do not have their own enzymatic pathway to produce nucleosides. They rely on cell machinery |
| What tissues is mitochondrial DNA depletion syndrome specific to? When does it affect the child? | Condition is very specific to muscle and neural tissue. Doesn't effect foetus - only affects child post-natally |
| What can cause microcephaly | Zika virus, mitochondiral DNA syndrome (disruptions to energy metabolism), foetal alcohol syndrome (disrupts neural metabolism) |
| What add weights to brain after birth | dendritogensis, synaptogenesis. myelination |
| different ares of the cortex blah and different times | develop! |
| What protein does the zika virus bind with? | The mushashi-1 protein, which binds RNA and is involved with cell proliferation |
| What are the three primary behavioural symptoms of autism spectrum disorder? | impaired social interaction, impaired social communication. restricted and repetitive motions |
| Is autism spectrum disorder highly heterogenous? | Yes but with a convergence on neural circuits and synaptic function |
| What is Rett's syndrome? When does it occur? What type of mutation causes it? | A severe type of autism spectrium disorder. Caused by random mutations (after fertilisation). Child starts of normal then they cannot speak, walk and have breathing problems |
| Describe the brain growth, size of cortical columns and balance between inhibition and excitation in those with autism spectrum disorders | Brain smaller at birth, then bigger postnatally then normal at adolescence (lots of children with autism grow out of it) They have a reduction in the width of cortical columns, which is determined by dendrites and synapses Too many inhibitory neurons |
| Where are the ASD susceptibility genes preferentially expressed? | Genes associated with transcriptional regulation and synaptic development are preferentially expressed in layers 2-4 in the cortex |
| What do layers 2 and 3 in the cortex do? | They project to the other layers and are involved in communication between brain regions |
| Describe the ability of people with ASD to transfer signals to different regionsof the cortex | It is impaired. Their sensory and motor interactions are impaired |
| What are the common symptoms of Rhett syndrome? | Ataxia, microcephaly, gradual loss of speech, seizures |
| What type of disorder is Rhett's disease? A mutation is what gene causes the disorder? | X linked dominant disorder. A mutation in the mecp2 gene. If present in male conceptus, the mutation is lethal |
| Is a mutation in Mecp2 always fatal for males? | No, not if they exhibit germline mosaicism. The mutation would have occcured in gonadal stem cells (arisen postnatally) --> proportion of his sperm contain the mutation. So he won't exhibit any symptoms but can pass the disease on to children. |
| Why are female carriers of the MECP2 gene exhibit highly variable phenotypes? | X-inactivation leads to mosaic expression of mutant MECP2 , therefore causing highly variable phenotype |
| What does MECP2 do? | Affects ability of transcriptional machinery from interacting with DNA (gene expression regulator) |
| Why does the Rhett phenotype rise postnatally | perhaps MePC2 controls gene expresion of dendritogenesis and synaptogenesis. Post natal cortex is susceptible due to massive growth occuring at this stage |
| Describe the dendrites of pyramidal neurons in cortex layer 3 in people with rhetts | they are shorter! L3 projects into other regions of the cortex |
| Different cortical neurons exhibit peak dendritogenesis at what? | different times! |
| Do neurons die in Rett Syndrome? | No what does this mean? Suggests that it is a neurodegenerative disorder that may be reversed |
| How did they reverse Rett's syndrome in mice? part 1 | Created a transgenic mouse which had a cassette containing a stop codon and two lox p sites inserted into MECP2 intron. Mice developed rett symptoms 6 weeks later. Mice were crossed with transgenic mouse, that had a modified eostrogen receptor |
| How did they reverse Rett's syndrome in mice? part 2 | The oestrogen receptor was coupled to Cre recombinase, which cleaves lox p sites. oestrogen does not recognise ostrogen, only binds to tamoxifen. When fed T, receptor translocates to nucleus to affect gene expression |
| In the experiment where they reversed the symptoms of Rett via LOX P and tamoxifen, where the symptoms permanent? | yes! |
| How do yo move towards a therapeutic approach to treating Rett? | Need to determine the genes/proteins that MEPC2 control s |
| What id BDNF? How is related to the disease progression of MECP2 | Brain derived growth factor. MECP2 controls its gene expression. Transgenic mice with a mutation in MECP2 crossed with mice with a BDNF gain of function mutation, rescued the phenotype |
| What processess does BDNF control? | It controls neuronal differentiation and growth, synapse formation and plasticity (particularly in glutamatergic neurons) |
| Describe the transportation of BDNF | Can act on post-synaptic cell plus it can act in an autocrine fashion. Acts on cell body and dendrites. Essentially one neuron can affect its own activity and the activity of others. |
| What happen when they selectively overexpressed BDNF in mutant MECP2 null neurons and WT neurons | Null = phenotype was rescued (dendritic morphology increased) WT = no rescue. In mosaic brain, mutant neurons need to be fixed due to the autocrine function of BDNF |
| Can an overexpression of MECP2 cause retts | Yes both over and underexpression can cause retts. |
| Are mice good models of social and cognitive behaviours? | No monkeys are better |
| Is it easier to knock out or over-express something in primates? | Over-express! |
| How did they over-express MECP2 in primates to induce Rett syndrome | Took lenti virus (which can take a lot of foreign DNA), which contained MECP2 (tagged with HA (antibodies identify it) and mCherry and GFP). They fertilised egg and injected virus so embryo would be affected. |
| When you transfect an embryo with a gene, where do you want it located | far away from exons so it doesn't effect other genes |
| What can be used to induce autism in mice? | Valproic acid |
| How did an enriched environment affect mice with and without autism | EE in mice with autism did not significantly reduce their anxiety or significantly increase their dendritic spine density. However, the mice with autism in EE experienced significantly reduced anxiety and increased dsd. It returned their levels to normal |
| How does neural activity affect gene transcription? | Glutamate binds to NDMA receptor. Causes Na+ and Ca2+ influx, which leads to the gene expression of intermediate early genes. |
| brain development is controlled both by | self-regulating cascades of gene expression as well as by experience (environment). |
| How does increased tactile stimulation affect the no. of dendritic spines, dendritic length and no. of bifurcations in normal mice | Increases them! |
| How does early life stress effect gene expression | Stress changes neural activity on brain. Hypothalamus releases vasopressin, which acts on anterior pituitary . AP releases hormones which act on adrenal glands to release corticosterone, which acts on brain to alter gene expression |
| How does high maternal stimulation affect MECP2 and vasopressin | It increases the methylation of CPG (regulatory region of vasopressin gene). Enables MECP2 to bind and downregulate vasopressin. (reduces corticosterone) |
| How does low maternal stimulation affect MECP2 and vasopressin | low maternal stimulation, reduces CPG methylation and allows higher expression of AVP, which leads to increased cortisol |
| what happens to dendritic spines when there is no stimulation | Spine is lost |
| How does stress affect spine density | High stress reduces spine density |
| If MECP2 is involved in something what else may be involved | BDNF! |
| What are the critical regulators of dendritic spine development? How do they interact? How much of each is required for neuronal maintenance, synaptic | glucocorticoids and BDNF. High BDNF and low GC levels are required. Link between GC and BDNF is complex, sometimes GCs may directly stimulate BDNF |
| How does environmental enrichment affect BDNF levels in MECP2 null mice? How does it affect anxiety? | It significantly increases BDNF levels and reduces anxiety |
| Suggest a strategy using transgenic mice to overcome the early lethality of ubiquitous loss of BDNF part 1 | Need to create KO BDNF postnatally in cortex Create conditional mutation by LOXP, which can be recognised by Cre Transfect lox p into embryonic stem cell from blastocyst via homologous recombination |
| Suggest a strategy using transgenic mice to overcome the early lethality of ubiquitous loss of BDNF part 2 | Inject stem cells into blastocyst --> chimera -->mosaic germline -- cross chimeras to produce complete BDNF knockout |
| Strategy: use transgenic mice to overcome the early lethality of ubiquitous What is needed to make this strategy work? | Need to create a line of transgenic mice expressing cre recombinase ONLY in post-natal mie around 3-4 weeks. Use CAMK promotor (which is expressed around that time). Or use modified eostrogen receptor (tamoxifen) |
| What do you expect to happen if you cross these mice against the MeCP2 mutant mice | need to know effect MECP2 null has on BDNF decreases spine density |
| What happens when in BDNF and MECP2 KO mouse | Complete loss of BDNF enhances the phenotype |
| Why can't axons regenerate in the CNS system? | Myelin contains inhibitory molecules which prevent them from growing. |
| What are some possible reasons why human stem cells exhibited extensive neuronal growth in rats | Maybe inhibitory rat molecules cannot bind to human receptors. Maybe humans do not express necessary receptors. Perhaps timing is off |
| Describe the major components of a growth cone | Dense core- comprised of tubulin, vesicles mitochondria and microtubules Lamellipodia - filapodia |
| What are the five major principles of axon growth? Part 1 | 1. Axons need an adhesive surface to grow (do not grow in fluid) 2. Growth cones do not contract and drag axons like fibroblasts 3. Axons grow in straight lines unless instructed otherwise |
| What are the five major principles of axon growth? Part 2 | 4. Neuronal differentiation produces basal axon outgrowth which requires stimulation by exogenous factors 5. Growth cone and axon morphology is dynamic |
| What types of surfaces do axons grow on and why | Collagen - adhesive due to receptors that bind to neurons Poly-L-Lysine - positively charged - electrostatic interactions |
| Describe the general relationship between adhesion and axon growth | Bell curve - too little adhesion - no growth, to much - no growth |
| What are the three different types components that can provide adhesion in axon growth | Strength of adhesion between cell surface molecule and substrate, strength of interaction of signalling units, strength of interaction cytoskeleton and cell surface molecule |
| What are the receptors which bind to laminin in collagen (ECM) | integrins! |
| What links integrin to the cytoskeleton | whole series of bridging molecules |
| What does the multiple forms of integrin and laminina mean? | Provides different adhesion strengths between integrins and lamina |
| How can guidance cue receptors affect integrins | Guidance cue binds to receptor, which affects kinases. Kinases change conformation of integrin ,results in signalling, activation of cytplasmic proteina and actin binding |
| Describe the fish regeneration experiment part 1 | Analysed the axon growth in fish retinal ganglia Placed ganglia on mosaic of lysine + laminin and myeoin Avoid myelin and grew on lysine + laminin |
| Describe the fish regeneration experiment part 2 (conclusions) | Therefore fish express receptors that are capable of interacting with inhibitory molecules. Fish myelin must not express sufficient amounts of inhibitory molecules to stop axon growth. HEnce why fish myelin regenerates |
| How do growth cones move? | Plasma advances along or between filapodia Central region of organelle is pused forward Conversion of old grwoth con into new axon |
| Describe how actin dynamics drive filapodia movements (basic actin info) | Actin has polarity. Polymersied at distal end and degraded at proximal end. Degraded actin used for polymerisation. |
| Describe how actin dynamics drive filapodia movements (role of myosin motors) | Myosin motors forces actin to undergo actin flow. Therefore if there is a balance between polymerisation and degradation, filapodia retract. Motors between actin and microtubules provide tension - directs microtubule growt towards site of adhesion. |
| How would you manipulate the system to promote axon growth | Enhance the enzymes that control polymerisation Reduce the enzymes that control degradation Inhibit myosin motors |
| What happens to the myosin motors if adhesion is too tight | Tight - slow retrograde flow. Too tight - Myosin motors cannot overcome strength |
| What controls tubulin growth | Adhesion results in axon and tubulin growth. Tension via myosin motors and actin enhances tubulin polymerisation. Enzymes which control tubulin growth respond to force |
| Jasp blocks myosin activity? What happens to growth cone | No retrograde flow, no actin turnover for polymerisation. No growth |
| Myosin 1 and II control | treadmilling |
| Describe the phophorylation pathway of myosin ii | Receptor is activated. Activates the g protein Rho, which interun activates ROCK (rho cytoplasmic kinse), which phosphorylates myosin II, leads to axon retraction |
| How do GAPS and GEFs affect Rho activity? | Gaps - GTPase activating protein. Enhances GTP -> GDP. Rho inhibits itself faster GEFS - removes GDP and allows GTP to bind. It activates Rho |
| How do Rho and Rock inhibitors affect spinal cord repair | Help block neural retraction and increase spinal regeneration |
| What is c3 and Y? | Rho inhibitor |
| What happens to microtubules when filapodia hits target | So filapodia are constantly treadmilling - sensing their environment. When one hits a target and interacts with receptor, microtubules bend towards target |
| What are the g proteins that stimulate elongation (GEFs) | CDC42 and RAC |
| Describe how DCC and netrin work to induce axon guidance/elongation | High concentrations of netrin bind to DCC receptors in growth cone cytplasm. Causes dimerisation of DCC, enables it to bind to CDC42 and RAC. Causes interaction between binding protein and rac. Grow towards nectin |
| How does netrin affect actin | Reduces retrograde flow |
| What did the hippocampal neurons on cultured PLL and Ln reveal? | Numerous processes extend and retract. Only becomes an axon when they touch LN. Therefore, substrate is important even when all the growth factors are present. |
| The amount of growth is dependent on what in the environment | Amount of adhesion molecules in the environment |
| What are the main cell adhesive molecules? What experiment did they perform to determine its role? | N-Cams. Took lipid molecules expressing n-cam and determined whether they became attracted to each other.+ did experiment where they used anti-bodies to block it THey did, therefore homophilic binding. |
| What are the main chemotropism molecules | Netrin + DCC |
| What are the main chemotropism molecules | Slit/ROBO |
| Is DCC purely chemoattractive? | No, in presence of UNC-5 DCC is repulsive. Activates Rho pathway |
| Describe the journey of the dorsal commissural neurons? | Start in ourter edges, whih contains ECM and integrins. Then they grow ventrally (but avoid motor neurons) and cross the midline at the ventral commissure. Then they grow caudally or rostrally |
| Describe the ventral area of the spinal cord in terms of cell types | Contains the floor plate which contain a special kind of glial cells that have lateral processes. These processes provide substrate for axons growing across the midline. |
| Describe the ventral area of the spinal cord in terms of cell types | Floor plate cells. No lateral processes. No axons cross midline here |
| What was the major molecule in the ventral floorplate of the spinal cord attracting the axons to the midline? How did they determine this? What other conclusions did they form? | Netrin 1 was acting a guidance cue. Placed dorsal part next to floor plate. Axons grew out. Netrin 1 KO had reduced ventral commisure. Therefore antoher chemoattractive molecule must be present |
| How did they test for residual activity in the floor plate? What other chemoattractive molecule was present in the floorplate. WHat is its role outside of the floorplate and what is its receptor? | Express floorplate ectopically in netrin 1 KO. Axons still grow ventrally, SHH was the attractive guiding cue. Acts on BOC receptor. Diffuses from notochord into spinal cord to act as motor neuron differentiator |
| How did they determine that netrin 1 was epressed outside of the floorplate | FLoorplate KO, Axons still crossed midline towards v3 interneurons. These neurons express netrin 1 |
| What are the molecules that mediate chemorepulsion in the dorsal spinal cord | Draxin through DCC BMP7 through BMP1 and 2 GDF7 |
| Is netrin purely chemoattractive | no in the presence of UNC-5, netrin and DCC are chemorepulsive |
| What are the molecules preventing the axons crossing the midline of the spinal cord more than once? | Roundabout receptors (ROBO) and their ligand slit. |
| Describe the neurons that express ROBO | They show a ROBO gradient. Once their axons cross the midline, ROBO is upregulated. |
| What Robos are expressed in the spinal cord? What happpens when you KO robo 3? What happens when you KO both Robo1 and RObo 3 | 1 and 3. KO of 3 leads to a similar phenotype of netrin 1 KO. Axons do not make it to the midline. KO 3 and 1 - axons reach midline and some axons cross |
| What did they conclude from the KO studies of Robo 1 and 3? | Robo 3 must interact with RObo 1. Robo3 inhibits Robo 1 allowing axons to cross midline |
| Describe the model of axon crossing part 1 | Attraction to midline - netrin activation of DCC. Robo 3 inhibits 1 prior to interaction with slit (slit expressed at midline) INteraction between DCC and ROBO silences DCC. Axons cross. |
| Describe the model of axon crossing part 2 | Robo becomes responsive to slit at midline, therefore axons cannot cross back. Mechanism unknown, but may be to do with enhanced ROBO concentration |
| What is a stem cell? | A cell that both self renew and also give rise to many undifferentiated progeny |
| What are the four major cardinal features of a stem cell? | Self renewal for an EXTENDED period of time, proliferation, differentiation, regeneration of tissue following injury |
| What are the types of division that stem cells can undergo? | Cell death - on eof the cells dies - just self renewal Symmetric - both the same cell i.e. both stem ells Asymmetric - one stem cell, one progenitor cells |
| What are the two major issues associated with identifying stem cells? | 1. Stem cell phenotype changes throughout development 2. Stem cell share attributes with other cel types |
| What are the three major ways you can identify stem cells? | Positional cues, in vitro assays and FACS |
| Describe how you can use positional cues to identify stem cells. | Stem cells replicate. Can use BrdU, which is taken up in replicating DNA and then use antibodies for identification. Lgr5 is a g-coupled receptor expressed specifically in stem cells. |
| Which is more common asymmetrical or symmetrical stem cell division? | assymetric |
| Which occurs first differentiation or maturation | Differentiation then maturation |
| Can progenitor cells proliferate and give rise a wide range of cell types | They can proliferate but they only give rise to limited cell types |
| What is the most rapidly regenerative part of the body? | Intestinal epithelium |
| How could you definitely demonstrate that Lgr5 positive cells are stem cells. | Create reporter mouse. Took promotor of lgr5 and attached it to marker. See if stem cells proliferate. See if they can repopulate an area after injury/ablation. SC have a unique envrionment - identify ultrastructural characteristics |
| Where are adult stem cells found in the brain? | Subventricular zone |
| Do adult stem cells in the brain divide often? | No they are mostly quiescent (more divisions, more opportunity for mutations to occur) |
| How could you ablate a tissue to see if stem cells are present in the sub-ventricular zone | Give them anti-mitotic drugs. Kill other cells not stem cells as they are quiescent in the brain |
| Describe how you can use in vitro assays to identify stem cells | Take cells from subventricular zone and culture it. Promote their proliferation into neuro-spheres via EGF. Remove EGF to promote differentiation |
| Describe how you can use FAS (fluorescent activated cell sorting) to identify stem cells. Do stem cells tend to have positive idenitification markers? | Sorts cells based on propoerties. Stem cells tend to be larger. Expression of lrg5 (fluorescent anti-body) No so use markers from non stem cells as an exclusion criteria. After isolation, culture it to make sure |
| What are the major limitations of the current stem cell identification methods? | Stem cells are removed from their normal context Cell no. low from onset Growth factors promote proliferation at expense of differentiation Larger spheres have nutrient absorption contraints for FACS; how do you markers are unique to sc |
| Where are developing neural stem cells located? | Neuroepithelial cells/radial glia are found lining the ventricles throughout the neuraxis. Give rise to CNS. Within the cortex, secondary pool of progenitors is found in SVZ |
| Where are adult neural stem cells located? | Cortical neural stem cells are found in the SVZ lining the lateral ventricles (olfactory bulb source) and the subgranular zone of hippocampal dentate gyrus |
| What is a neurogenic nice? WHat important features are present in such a niche? | An area that supports ongoing neurogenesis. Vasculature, CSF, ECM, contact between cells within the niche are all important in preventing early differentiation and maintaining quiescence |
| Do stem cells directly differentiate into neurons? | No they produce intermediate progenitor cells, which differentiate into neurons and migrate up the neuroglia scaffold |
| After formation of cortex, stem cells turn into what | astrocytes |
| Stem cells always maintain contact with what | ventricular and apical surface |
| What type of division do NSCs undergo in the ventricular zone (symmetry and orientation of cleavage) | VZ = early - symmetric - self renewal. Late - assymetric (NSC + daughter). They almost entirely divide by vertical cleavage |
| What is the significance of vertical cleavage in radial glial cells? | Postulated that vertical cleavage enables both daughter cells to inherit part of the membrane surrounding the cilia of the cell |
| What type of division do NSCs undergo in the sub ventricular ventricular zone (symmetry and orientation of cleavage) | Progenitors undergo symmetric division along horizontal cleavage plain |
| What does adult neurogenesis regulate? | Learning and memory and innate olfactory responses (predators, mother-child, pheromones) |
| What are some sources of NCS for use in disease modelling/therapy | embryonic stem cells, reprogrammed cells, endogenous adult neural stem cells. |
| What are some disease therapies stem cells could be used for? | Spinal cord injury, parkinsons, neurdegenerativ e diseases |
| What are the pros and cons of embryonic stem cells | Cons - source of cells is ethically contentious. Pro - can potentially provide any neural or neuronal cell type in the brain |
| What are some pros and cons of using fetal stem cells | Cons - source is often aborted fetuses and may contain genetic abnormalities Pros: well characterised and easily derived |
| What are some pros and cons of using adult endogenous stem cells | Cons: NCS are likely regionally specified and less potent when transplanted to another area Pros - no ethical issues |
| Overall what are the pros of using IPSCs | No ethical considerations Autologous cells overcomes immune system rejection issues |
| What are the cons of generating IPSCs via virus transfection | Random integration of virus - mutations Reactivation of virus very inefficient |
| What are the pros and cons of generating IPSCs via recombinant proteins | Pros - no virus, no random mutagensis Cons: inefficient,•Requires prolonged passaging, which is associated with karyotypic abnormalities |
| What are the pros and cons of generating IPSCs via transposons? | Pros: MOre efficient than virus, transposon is excised out after cell generation so no mutagenesis. Cons: Requires prolonged passaging, causes increased CNVs, may retain part of previous gene expression signature (regulatory stuff), full epigenetic re? |
| What are some basic problems associated with transplanting stem cells | Tumour potential Immune rejection |
| What are some questions you need to consider when transplanting stem cells for spinal cord injuries | Where to transplant Timing - when - need to avoid glial scarring and immune system |
| How can we increase endogenous neurogenesis | Exercise, environmental enrichment |
| What converts ectodermal tissue into the neural tube> | Sonic hedge hog! (gradients of morphogen) |
| In which directions is the neural tube pattenered? | Ventral-dorsal and rostral-caudal |
| What happened when they ectopically expressed the notochord | Another muslce plate formed. Therefore the notochord is involved in the specification of floorplate and motor neurons |
| In which direction does the roofplate of the neural tube strech out in? What implications does this have for the alar and basal plate formation? | It stretches out rostrally. |
| Where does the neural crest originate from? What does it give rise to? | Originates from the notochord and it gives rise to the peripheral nervous system. |
| The notochord makes tissue what | Makes tissue nervous tissue |
| What is responsible for the patterning in the hindbrain (rhombocephalon) | The hindbrain rhombomeres are patterened by Hox genes in the anterior posterior axis (TF). Gene expression is specific to a particular rhombomere (selective gene expression) |
| What are the branchial arches? Where are they located | They arere a series of externally visible anterior tissue bands lying under the early brain that give rise to the structures of the head and neck. In the hindbrain! |
| How many rhombomeres can the hindbrain (rhombocephalon) be divided into? | 8! |
| How does the human face develop? | Neural crest cells migrate down to form the different parts of the face. |
| Facial deformities are caused by what | problems with the neural crest |
| What is the boundary between the midbrain and hindbrain called? What is its purpose in terms of development. | Isthmus - major signalling centre. Organising region. |
| What morphogens are secreted at the isthmus to impart identity areas of the midbrain and hindbrain | Shh FGF-8 Wnt-1 |
| How does sonic hedge hog operate on a molecular level to impart identity/ | Gradients of shh activate receptors, which in turn activate transcription factors |
| What are the two transcription factors that impart identity to the hindbrain and midbrain boundary (isthmusz0 | Otx-2 Gbx-2 |
| What are the official anotomical names for the midbrain and hindbrain | Midbrain - mesencephalon Hindbrain - rhomboencephalon (mentencephalon - anterior, mylencephalon - posterior) |
| What evidence is there that the isthmus is a major organising region? | Transplantation of the isthmus can induce tissue to form an ectopic midbrain and cerebellum. Ectopic expression of FGF8 led to development of 2 cerebellums |
| How is the cortex patterened? | Not patterend via defined TF gene expression . Patterened via morphogen gradients |
| What molecules are required for the expression of the cerebellum and and the superior colliculus? | Cerebellum - FGF8 Superior colliculus - engrailed (maybe bit of Wnt-1) |
| How is the forebrain patterned? | Partitioned into different prosomeres based on the expression of different tracription factors. |
| What are the two ways that patterning occurs? | Occurs through defined expression of TF (e.g. rhombomeres) or though concentration gradient of morphogens (which alter gene expression of TF) |
| What is an area of the cortex defined by? | Defined by inputs and outputs |
| How is axon projection regulated | First by patterning then by activity |
| What are the organising regions of the telencephalon (cerebral cortex) | Commissural plate cortical hem - gradients of morphogens |
| Other than morphogens, what other molecules are expressed in gradients across the cortex? | Transcription factors! |
| What molecule is expressed at the telencephalic midline and is controls area position and identify in the cortex | FGF8 controls the expression of the four transcription factors, which imparts arial identity to the cortex |
| The barrel cortex system (somatosensory system) is evidence of what | evidence that brain processes info in an ordered manner. |
| Describe the rat barrel cortex system? | Whiskers arranged in specific rows. Arrangment is maintained in the cortex in the cortex. Whisker corresponds to a barrel |
| What happened to the barrel cortex system when they pulled the whiskers out? | Cells were still there but they were randomly dispersed. Discrete barrels did not form |
| Describe electroporation | Introduction of a plasmid via electroporation during a specific place and time during development. As DNA is charged, it is drawn in |
| What are the four TF factors that impart arial identity to cortex and what do they do? | Emx2, Pax6, Coup-TF1, Sp8 EMx2 - visual field Pax 6 - somatosensory Coup-TF1 - motor Sp8 - motor |
| What did the FGF8 studies show by Grove? | FGF8 represses Emx2 and Coup-TF1. Overexpression rostrally (normal position) moves barrels caudally. Overexpression caudally = duplicate barrels. |
| What do the EMX 2 studies by O' leary show? | EMX2 gradient of expression is important for defining cortical areas. |
| What happened to the size of the motor, visual and somatosensory cortexes when EMX2 was KO | Everything moved caudally. Visual centre decreased. Motor and somatosensory increased in size |
| What happened to the size of the motor, visual and somatosensory cortexes when EMX2 was overexpressed | Everything moves rostrally. Visual centre increases in size, motor and somatosensory decrease |
| What happened to the size of the motor, visual and somatosensory area in a Coup-TF1 KO | Motor increases and moves caudally, the rest decrease in size |
| What happened to the size of the motor, visual and somatosensory areas in a Sp8 KO | Motor moved rostrally and decreases in size. The rest moved up rostrally. Somatosensory decreases in size and visual increases |
| Can changing cortical areas influence thalamic patterning (Is there top-down plasticity in the system?) | Yes - barreloids was smaller in thalamus.THalamic row A die - no space for them Barellettes normal - therefore effect is only 1level |
| Describe the experiment where they disrupted the whiskpers and some projected ipsilaterally. How did this alter brain function? | So a ROBO3 KO mouse resulted in some ipsilateral projections. inputs from both sides in both hemispheres - the mutants have trouble with object discrimination test . So somatosensory ability was impaired |
| What are the common symptoms of people with agenesis of the corpus callosum and/or congenital mirror movement disorder? | Patients experience deficits in sensory and motor function, language development, social interaction |
| Do callosal axons cross the interhemispheric fissure? | No! |
| What remodels the intehemispheric fissure? And what shape do they differentiate into? What does this allow? | Radial glial remodel it into integrated tissue! They differentiate into multipolar astrocytes called midline zipper glia. The fusion of these glial faciliate the formation of the corpus callosum. |
| What happens to the glial differentiation and corpus callosum formation when you KO and overexpress FGF8 | KO - delayed differentiation - no fusion, no bridge, probst bundles form Excess - precocious differentiation - probst bundles |
| Through what molecule does FGF8 mediate interhemispheric remodelling? How did they determine this? | NF1 KO mice - over expression of FGF8 we get immature glial forming but no mature. Therefore FGF8 is controlling glial regulation via NF1 |
| Does interhemispheric modelling occur in marsupials or monotremes? | No! Their brain is connected via the anterior commissure |
| How do DCC contribute to interhemispheric remodelling? | DCC not only involved in guidance but also in glial differentiation DCC induces actin rearrangement in glia, facilitating their differentiation. |
| What type of mutations were found with ACC and congenital mirror movement disorder? | DCC! |
| DCC and Netrin1 knockout mice display defects in | midline remodelling |
| what happens to Midline zipper glia in DCC knockout mice? | They remain bipolar |
| What is neuronal specificity? | The ability of neurons to connect with a selective number of targets in the brain |
| Is there a lot of redundancy in the axon guidance system? | Yes! |
| Short range guidance/recognition molecules mediate what type of interaction | Membrane bound cell surface proteins that mediate interactions between neurons therefore either homo or hetero |
| Long range guidance/recognition molecules mediate what type of interaction | Typically heterophillic - secreted ligand interacts with membrane bound receptor |
| What are the common domains in recognition molecules? And what are their functions? | Signal sequence - puts in secretory pathway Extracellular domain - interactions wtih other proteins and ECM Transmembrane - Cytoplasmic tail - signalling, interactions with cytoskeletons |
| Drosophila photoreceptors (R cells) can be used as a model for layer specific targeting. Layers R1-6 target what, while R7-8 bind to? | 1-6 - targetLamina 7-8 -target medulla |
| What type of molecule is Caps? What type of binding does it mediate? | Caps is a Leucine Rich Repeat (LRR) recognition molecule. In vitro - mediaters homophillic, in vivo uncertain. |
| What role does the recognition molecule Caps play in axon guidance in drosophila photoreceptors (R cells)? | Caps mediates layer specific targeting of R8 neurons. |
| What evidence was there that Caps mediates layer specific targeting of R8 neurons? | Expressed in R8 cells and R8 target layer R8 cells make targeting errors in caps mutants R7 cells force to express Caps target to the R8 layer |
| Is Caps autonomously required by R8 cells? | Yes, R8 cells need to express Caps on cell surface to target correctly |
| What is required for R7 targeting? | N-cadherin is autonomously required in R7 |
| What role does N-Cadherin play in axon guidance in drosophila photoreceptors (R cells) What type of binding does it mediate? Is it autonomously required? Is it broadly expressed in the visual system? | Required for R7 neuron targeting, .Mediates homophillic binding. Yes it is autonomously required. Yes broadly expressed. |
| Yow can a protein expressed everywhere specify neural connections | Gradients - differences in level of expression Specificity comes from another ligand/co receptor - redundancy Timing - temperol differences between neurons |
| What is a permissive cue? WHat is an instructive cue? | P - Doesn't contain specific info, simply acts like a glue (adhesion). I - Lock and key mechanism that induces synapse formation. Requires signal transduction |
| What dictates the organisation of lamina cartridges in the fly visual system when all of the R and L cells express N cadherin | High levels of N cad are expressed at core of cartridge (L1 and L2 cells), whereas lower levels of Ncad are expressed by cells in periphery (R1-5 and L-3-5). Differential expression regulates position within the cartridge |
| What evidence was there that differential N cadherin expression was responsible for controlling the organistion of the lamina cartridges within the fly visual system? | Manipulating Ncad levels in L and R cells changes cartridge organisation Overexpression of Ncad in outer L cells causes them to move inside of the cartridge |
| How could you determine whether Ncad is permissive or instructive? | Rescue with Ncad with no signalling capabilities (no cytoplasmic tail) Rescue = permissive |
| How could you determine whether Caps mediates homophillic binding? | Express Caps in R8 when the rest of the animal is mutant for caps. Rescue = homophillic binding is not required. No rescue = homophillic binding required |
| Why are hippocampus synapses easy to visualise? | They are large and have a multiple vesicle release sites |
| How did they recapitulate the synaptic specificity of mossy fibre synapses? Part 1 | Microisland Assay: Coat slip with agarose Plated PDL/collagen onto dish to provide ECM for cells to grow on Plated astrocytes, then CA1 and CA3 neurons in limited conc Then plated DG cells with GFP and pre-synaptic marker |
| What did they conclude form the mossy fibre microisland assay in terms of which neurons DG was attracted to. How did they measure this | DG - C1 = repulsion DG- C3 - adhesion Measured punkte, strength of transmission (amp of AP) |
| When you are using anti-bodies to identify synapses what do you need to do | Use a whole lot of anti-bodies so you can create different colours for each factor. |
| What molecule is required for DG-CA3 synapse formation? | Cadherin-9! |
| How did they determine that cadherin-9 is required for DG-CA3 synapse formation/what evidence is there? | CAD-9 KO had reduction in DG CA3 synapses compared to control. Cad9 rescue (cad9 that was resistant to iRNA) had same no. of synapses as control. KO caused dendritic defects in CA3 cells |
| What is a simple system for studying neural connectivity? | C.elegans vulva! Neurons develop to contract vulva |
| How are synaptic connections specified in the C.elegans vulva? | HSNL synapses on both vulval muscles and VC (VC 4 and 5) neurons. |
| What happens to pre-synaptic localisation in the C.elegans vulva when the muscles and VC neurons (post-synaptic targets) are ablated? | Still had pre-synaptic specialisation. |
| How could pre-synaptic specialisations form in C.elegans vulva in the absence of target cells (mulsce and VC neurons) | Guidpost cells! |
| What are guidepost cells? | A cell that induces synaptic organisation but it is not involved in the synapse. Similar to intermediate targets in axon guidance. |
| What are the guidepost cells for HSNL in C. elgans vulva? How did they determine this? | Primary vulva epithelial cells! Mutant with no vulva epithelium had pre-synaptic localisation in incorrect place. Mutant with multiple vulvas and epithelial had multiple pre-synaptic localisations. |
| How do guidepost cells induce localisation of synaptic vessels? | Through proteins (recognition molecules) expressed on cell surface. |
| How did researchers determine what recognition molecule on the vulva primary epithelial cells was responsible for mediating the formation of HSNL synapses | Used a genetic screen - EMS screen and searched for mutants which had defects in pre-synaptic localisation (punkte were mislocalised). Worms were expressing synaptically localised GFP IN HSNLS |
| What recognition molecule on primary epithelial cells ids required for HSNL synaptic localisation. What is its ligand and the function of its ligand? | SYG-1 and SYG-2 is its ligand. 2 - is required for HSNL synaptic vesicle localisation. |
| How did they find out where SYG1 and 2 were expressed. Where were these molecules expressed? | Made GFP fusion proteins between SYG1 and SYG 2. Found that SYG2 was localised to primary epithelial and SYG1 is localised to HSNL. |
| What happens when you express SYG2 in secondary epithelial cells? | It mislocalises SYG-1::GFP (couldn't do KO in pirmary epithelial cells to prove molecule is autonomously required) |
| Describe the model for how SYG-1 and SYG-2 specify HSN synapses | HSNL expresses SYG 1, which interacts with SYG 2 on primary epithelial cells. THrough heterophillic binding and signalling you acheive pre-synaptic specialisation, which allows these neurons to form synapses with muscles and VC neurons |
| What type of interaction is important for permissive cues? | Repulsive! |
| Describe semaphorins (Sema3E) and their receptors plexin (PlexnD1) and neuropilin (Nrp). What type of interaction do they mediate. | Sema3E is a secreted ligand. When it binds to the receptor PlexnD1 it mediates repulsion. When it binds to the receptor PlexnD1 in the presence of the co-receptor Nrp it mediates attraction |
| Whether a receptor mediates adhesion or repulsion is dependent on | co-receptors and intracellular environment |
| Describe the muscle reflex circuit | Sensory neuron synapses with and excites motor neurons in spinal cord. Also excites spinal interneuron, which inhibits motor neuron to flexor muscle. Motor neuron escites muscle fibres and flexor muscles relax |
| How can you distinguish between a monosynaptic vs polysynaptic reflex arcs | Monosynaptic circuit (e.g. triceps) produce APs faster than multi-synaptic (cutaneous maximus). Can measure using electrophysiology |
| Why did researchers wonder whether Sema3E prevented circuit from being monosynaptic | There was differential expression of Sema3E in monosynaptic and multi-synaptic circuits |
| How did they prove that Sema3E was sufficient in preventing mono-synaptic circuits? | Sema3E KO - connections within sensory afferent MN circuit became mono PlexinD1 KO - contacts became mono When Sema3E was ectopically expressed in triceps MN =, the number of mono contacts severly reduced |
| What prevents triceps proprioceptor from making poly synaptic connection? | Triceps do not express SemaE3 which converts mono -> poly |
| Do Sema3E and PlexnD1 affect motor pool specificity? | No! The circuit remain intact, but additional synapses are added (proprioceptor attaches to specific interneurons) that make the circuit longer. |
| Repulsive cues can provide what type info? | Instructive information to the wiring of neural circuits |
| What do Dscam molecule mediate? | Homophillic binding that leads to repulsion |
| What are the two types of homophillic repulsion? And which Dscam mediates each type? | Repulsion between different neurons -> dscam 2 Repulsion between processes from the same cell (self avoidance) -> Dscam1 and Dscam2 |
| Describe the specificity or photoreceptor synapses (laminar cartridge of the fly visual system) | Multiple (4) post-synaptic targets for photoreceptor synapdes. Each with dendrites from L1 and L2. L1 detects light, whilst L2 detects dark edges. |
| How did they determine that self avoidance regulates photoreceptor synaptic specificity? What evidence was there that suggested that Dscam 1 and 2 specify L1-L2 post-synaptic pairing? | Flies mutant for either Dscam1 and Dscam2 had minor synaptic defects. Double mutants had significant synpatic defects. L1 and L2 processes were randomised . (L1 interacted with L1 and L2 with L2, whereas WT had no L1-1 2-2 pairing. |
| Both L1 and L2 express Dscam1 and 2 but repulsion only occurs between processes of the same cell. How is this possible? Why do L1 and L2 not repel each other? Part 1 | Alternative splicing generates f isoforms, which differ in extracellular domains. Each neuron has a unique Dscam identity. Isoforms will only bind if they are the same. - isoform specific homophilic binding |
| Both L1 and L2 express Dscam1 and 2 but repulsion only occurs between processes of the same cell. How is this possible? Why do L1 and L2 not repel each other? Part 2 | L1 and L2 express different Dscam2 isoforms. Therefore cannot mediate repulsion between different neurons, only between sister dendrites. |
| How many isoforms do Dscam1 and Dscam2 have? | Dscam1 - thousands Dscam 2- only A or b. Isoform A only binds with A |
| What happens when L1 and L2 are forced to express the same Dscam2 isoform? | 20-30% reduction in synapses (punkta). You would expect no synapses, but there are some indicating that there are back up mechanisms (redunancy in the system) |
| Describe the basic characteristics of zebra fish. Why are they good models? part 1 | Small, generation time (3 months), male and female not genetically determined, large clutches (500 offspring), rapid external development, transparent through early life stages, brains that relate structurally/func to humans |
| What are benefits of Zebra fish being transparent | No dissections needed, observe tissue in live animals, time lapse, observation and manipulation of neural function in vivo, great for optogenetics |
| Which parts of the zebra fish brain do we share a strong homology with? | Sensory systems and hindbrain. First and second processing in all sensory modalities. Cerebellum (receives sensory, speech, co-ordinated movement) and medulla oblongata (breathing, digestion) |
| Which parts of the zebra fish brain do we share a moderate homology with? | Midbrain structures and most of forebrain. Higher order processing (non-cortical) Amygdala and hippocampus, tectum (superior colliculus), thalamus, hypothalamus |
| Which parts of the zebra fish brain do we share a shaky homology with? | Cerebrum - anything cortical in humans is represented very differently in fish. |
| Are zebra fishes easy to house, do they have big clutches, are they good models for genetic tools, do the have a short generation time, external development, transpart, similar to human circuits, good for modelling complex behaviours. | Easy to house, big clutch, good for trangenics, bad for KOs, long generation time, have external development, transparent in early life stages, similar to humans except for cerebrum, good for modelling complex behaviours |
| Define forward genetics | Starting with a phenotype and pursuing and explanation (gene and mechanism) |
| Define reverse genetics | Starting with a gene of interest and modifying it to produce a phenotype |
| What is essential for for forward genetic screen | A large clutch as mutations are rare |
| Describe the forward genetics experiment with the belladonna mutants. Part 1 | Wanted to analyse the retinol tectal system. Performed a forward genetic screen using the chemical mutagen ENS. ENS induced random germline mutation. Crossed hetero to form homo mutants. Mutants were called Belladonna and had a midline crossing problem. |
| The mutated belladonna gene encoded for what? What | Mutants had a homozygous mtuation in a Lim domain transcription factor. Consequently numerous important axon guidance signals are weakly expressed or mixexpressed. |
| Summarise the steps undertaken in a forward genetics screen test (for anatomy) | Choose a neural structure of interest Mutagenise and screen for mutants Identify gene that causes phenotype Characterise how loss of gene causes defects in nervous system |
| How many different mutants were there in the belladonna screen? | 114 |
| Describe the four different tests used to characterise behaviour in zebra fish for forward genetic screens. Part 1 - VBA | Visual Background Adaptation test: Measures whether zebra fish can correctly camouflage to their environment Tests for light detection only Few demands placed on other systems Few demands |
| Describe the four different tests used to characterise behaviour in zebra fish for forward genetic screens. Part 2 - OKR | Optokinetic Response: Tests for broad field motion detection (horizontal) Requires that motor control of eyes is normal |
| Describe the four different tests used to characterise behaviour in zebra fish for forward genetic screens. Part 3 - OMR | Optimotor Response: Tests for broad field motion detection (vertical) Requires swimming |
| Describe the four different tests used to characterise behaviour in zebra fish for forward genetic screens. Part 4 - Prey capture | Tests for motion detection and high acuity vision Requires swimming and fine motor control |
| Summarise the steps undertaken in a forward genetics screen test (for behaviour) part 1 | Choose behaviour of interest Mutagenise and screen for mutants Identify gene that causes phenotype Find an anotomical/cellular defect responsible for the behaviour Characterise how loss of gene causes structural defect |
| What are the benefits and drawbacks of performing a neural anatomy forward genetic screen | Simple and fast assays for phenotype Cellular phenotype is clear from the start Mutants may not have behavioural phenotype - study not that important |
| Does belladonna have a behavioural phenotype? | Yes! |
| What are the benefits and drawbacks of performing a behavioural forward genetic screen | Behavioural testing is slower and more involved. Cellular mechanisms must be found (if possible) Behavioural importance is guaranteed |
| What behavioural defects did the blumenkohl (blu) mutant possess? what did they hypothesise was wrong with the animal? | Poor performance in prey capture, particularly with small prey Basic OMR, OKR and VBA normal Therefore they hypothesised that there was a problem with visual acuity |
| What did the blu gene encode? | Encoded for a glutamate transporter, which loads glutamate at the axonal terminal |
| What was the structural phenotype in Blu mutants? Why does this phenotype cause defects in acuity? | Retinal ganglion cells (which are glutaminergic) do not provide as strong a glutamate signal to their post-synaptic partners. As the blu axon terminals release less neurotransmitter, they grow bigger (branch and a^2) to compensate. Acuity suffers |
| What is the problem with inducing legions to link behaviour and anatomy? | Doesn't allow you to address cell/circuit level function. You can infer function but do it does not provide info on specific neurons |
| What are the limitations of traditional anatomical and behavioural analyess | Anatomical - detail single cell morphologies but have not addressed function Behaviour - addressed functions of regions but not single cell types or their microcircuits |
| Describe the process of enhancer trapping | GFP gene incorporates into the genome (germline) It expresses GFP protein if it traps an enhancer GFP expression shows where the trapped enhancer drives expression Trapper keeps stable lines with interesting patterns |
| Describe the Gal4/UAS system | The Gal4 protein is expressed in the target region one animal, while another animal expresses the UAS::GFP transgene. When the animals are crossed, the GAl4 protein binds to UAS to drive the expression of GFP |
| Describe Gal4 Enhancer trapping part | Gal4 gene incorporates into the genome (germline) It expresses Gal4 protein if it traps an enhancer Gal4 drives expression of UAS-linked transgenes in the pattern of the trapped enhancer Trapper keeps interesting lines for croses with other UASlinked g |
| If you are describing behavioural circuits and are looking at the overall anatomy, what type of UAS system should you use? | UAS:GFP |
| If you are describing behavioural circuits and are trying to look at single neuron anatomy and to analyse what cells compose this region and what are their stuctures, what UAS system should you use? | UAS:VariegatedGFP |
| If you are describing behavioural circuits and are trying to obversive activity, manipulate activity and are tyring to determine what behaviours are tehse neurons involve with, what UAS system should you use? | UAS:indicator UAS:manipulator |
| Describe the variegated GFP UAS system | GAL4 protein is expressed throughout expression pattern Another UAS linked marker labels the whole expression pattern visually A variegated UAS:GFP causes expression of GFP in small subset of GAL-4 +ve cells |
| Why is GFP expression often variegated | Variegated expression due to inefficient UAS constructs or the transgene is inserted area under repressor elements |
| What is the purpose of UAS:indicator | Observe a neuron while they are performing a function |
| What are the benefits to using optogenetics in neuroscience? | Genetically encoded so can be targeted to specific cell types temporal control Broad yet specific control over neural circuits (look at entire cell population not just region or individual cells) |
| What can be used to detect neural activity? | GCaMP. Fusion between GFP and moieties from calmodullin. When bound by Ca2+, calmodullin structure changes, altering GFP barrel structure and leading to increase in fluorescence. |
| What are the four ways you can manipulate neuronal activity? | Kill cells, depolarise them, hyperpolarise them, block neurotransmitter |
| How can you kill genetically targeted cells? | Using Metronidazol. Mtz is a harmless prodrug but when applied to cell types expressing NTR gene it is converted to toxin in these cells. NTR is attached to UAS system. Gal4 expressed in desired cell thpes, crossed with NTR:UAS |
| How can you hyperpolarise genetically targeted cells? Does it test for necessity or sufficiency? | Transfect the Cl- channel Halorhodopsin. Once activated with light, hyperpolarisation will occur. Tests for necessity |
| How can you depolarise genetically targeted cells? Does this process test for necessity or sufficiency? | Via the transfection of Channelrhodopsin. Channel activated with blue light, leads to Na2+ influx and K+ efflux (depolarisation). Tests for sufficiency |
| What would be the control experiments for experiments using Channelrhodopsin? | Control = shine blue light on WT cells Control - shine different coloured light on mutants |
| What is is one method of activating targeted cells via neutransmitters. Does this process test for necessity or sufficiency? | Through the transfection of of light gated glutamate receptor. Light induces conformational change, facilitating binding of glutamate. Glutamate binding induces influx of Na2+ and Ca2+. Sufficiency |
| Describe a real world example of optogenetics. How did researchers determine what KA neurons do? Part 1 - what strains did they create, what were the behavioural outputs and how did they manipulate the neurons? | Strains: Gal4 in RB neurons Gal4 in MN and KA neurons Gal4 in KN Behaviour - forward swim and startle response Manipulation:LiGluR and tetanus toxin in Gal4+ve cells |
| Describe a real world example of optogenetics. How did researchers determine what KA neurons do? Part 2. What was the behavioural output when RB sensory neurons were activated by LiGluR? | Startle response |
| Describe a real world example of optogenetics. How did researchers determine what KA neurons do? Part 3. What was the behavioural output when both KA spinal neurons and spinal MN were activated by LiGluR. And when KA neurons were activated by themselves? | KA + MN -> forward swim KA -> forward swim |
| Describe a real world example of optogenetics. How did researchers determine what KA neurons do? Part 4. What was the behavioural output when both KA spinal neurons and spinal MN were killed by the toxin. And when KA neurons were activated by themselves? | KA + MN -> ablation of forward swim and startle response KA -> no effect on startle response, attenuation of forward response |
| Describe a real world example of optogenetics. How did researchers determine what KA neurons do? Part 5. What were the conclusions? | KA neurons sufficient in inducing forward swim response (LiGluR) and are necessary for forward swimmming (Tetanus toxin) |
| What are the five distinct processes in neuronal development? | Neuronal migration, neuronal polarity, axon growth and guidance, target recognition and synapse formation, maintenance and plasticity. |
| Why are C. elegans a good model system? | Small short life cycle (3 days) Invariable number of days amenable to genetics genome is sequenced single neuron resolution |
| What are three important applications using GFP? | Where a gene expressed (promotor fusion - replace coding region of gene with GFP) Where a protein is co-localised (gene tag, GFP attached to gene downstream of promotor) Visualise cells in vivo - gene tag - GFP attached to gene downstream of promotor |
| When they mapped the C.elegans genome from zygote to adult, what important thing did they discover? | Some cells died during development |
| C.elegans always have the same number of __ and ___ in the same way | same number of cells and develop in the same way -> provided insight into cell programmed death |
| When you create a GFP gene tag, what do you need to test? | Test chimeric protein is still functional. Create mutant and perform rescue with wild type gene tagged with GFP. No rescue = loss of function |
| How do C.elegans move? Mutations can cause a weird movement called | Sigmoidally. Some mutatns can roll around, this mutation is called roller |
| Describe the mechanosensory touch cells in C.elegans part 1 (ventral( | AVM - guided ventrally, when they reach ventral side axon guided anteriorly PVM - guided ventrally, when they reach ventral side axon guided anteriorly |
| Describe the mechanosensory touch cells in C.elegans part 2 (lateral) | ALM - axons do not go ventral, just lateral. Straight anterior with ventral branch PLM - Has two processes. Once anterior and posterior branch. |
| how can you visualise mechanosensory neurons | Insert GFP into coding region of a gene under the control of the Mec-4 promtor (which is a promotor important for mechanosensory neuron function) |
| Say you were intertested in finding out what contros the ventral guidance in C.elegans, How would you do it? | Find a mutant where ventral guidance is lacking. Forward genetics - randomly mutagenise animal and screen for correct mutant. Or reverse, select candidate genes and do a KO or RNAi experiment. Study phenotype and do rescues, sequence rescue gene |
| If the mutation is recessive will you see a defect in the f1 generation | no! Will see defect 25% of time in F2 |
| What is the dauer stage in C.elegans | Resistant stage during bad conditions. Stops eating, growing, |
| C.elegans mutants who have defects in dorso-ventral axis have mutations in what genes? What exactly is this mutation? | Unc-6 and Unc-40. THe AVM mechanosensory neuron does not reach ventral cord, instead it runs directly anterior to the head |
| What does a penetrance of 30% mean? | The phenotype is present in 30% of the organisms |
| How can you test whether two genes act in the same pathway (axon guidance - C.elegans) | Double mutants should have the same phenotype and an increased penetrance |
| Do unc6 and unc-40 act in the same pathway to control dorso-ventral axon guidance in C.elegans? | Yes they do. The defect of the double mutant unc-6 and unc-40 is not worse than the individual mutants. The penetrance of the double mutants was higher as well. Therefore they operate in the same genetic pathway |
| What type of molecule is Unc-6 (C.elegans) | A secreted molecule that is attractive. Controls ventral guidance of AVM. Homolog of netrin |
| How can you tell whether Unc-6 is an instructive or a permissive cue? | Ectopically express UNC-6. Choose promotor expressed dorsally to express UNC-6. If rescue = permissive, no rescue= instructive, phenotype should be worse as axon should be attracted dorsally |
| Is unc-6 and instructive or permissive cue? (C.elegans) | It is an instructive cue |
| What type of molecule is unc-40 (C.elegans) | UNC 40 is a transmembrane receptor for UNC-6. THese molcules are attractive cues, that mediate the ventral guidance of AVM . Homolog of DCC |
| How could you tell whether unc-40 is autonomously required to mediate the ventral guidance of AVM (C.elegans)? | Take a mutant for UNC40 and express WT copy UNC-40 selectively in AVM. Rescue: cell autonomous, No rescue - non cell autonomous |
| Is Unc-40 required cell autonomously? | yes it is |
| In unc-6 and unc-40 mutant animals only a proportion (about 30%) of the animals have a defective AVM axon that cannot reach the ventral cord.What is guiding the AVM axon in the 70% of the animals with a normal phenotype? How did they find these molec? | They performed another genetic screen and found the molecules slt-1 and its receptor sax-3(ROBO) induced the same defect as unc-6 and unc-40 mutations |
| Do slt-1 and sax-3 act in the same genetic pathway? What type of guidance molecules are slit-1 and sax-3? | Yes double mutant phenotype same as individual, penetrance increased. SLT-1 is expressed dorsally and Sax-3 is on the AVM axon, thereforethey mediate ventral guidance of AVM via repulsion |
| Do UNC-6 and SLT-1 act in parallel pathways in regulating AVM dorso-ventral axon guidance? | Double mutants (KO one molecule from each pathway) had stronger defects than the individual mutants. Therefore. UNC-6/UNC-40 and SLT-1/SAX-3 act in parallel to regulate the dorso-ventral guidance of the AVM axon |
| What controls the posterior-anterior axis of the AVM mechanosensory neuron? | Wnt molecules and their receptors Frizzleds. |
| How many Frizzled receptors/Wnt molecules do you need to KO to see an anterior guidance defect in AVM? Why is this? | Two. Double mutant analysis revealed that Wnt signals and Frizzled receptors regulate AVM anterior-posterior axonal guidance. Single mutants - no defect. Double - 25-30% penetrance |
| Where are the Wnt molecule EGL-20 and CWN-1 expressed? | Expressed posterior to AVM, therefore they may be acting as repellant cues |
| How could you test whether EGL-20 and CWN-1 are acting as repellant cues to mediate the anterior guidance of AVM? | You create EGL-20 and CWN-1 double mutants. Express EGL-20 anteriorly and posteriorly. Posteriorly should rescue phenotype, anteriorly should worsen phenotype if they are repellant |
| Through which receptors do EGL-20 and CWN-1 mediate their repulsive effect to guide the AVM axon anteriorly? | The MIG-1 and MOM-5 receptor |
| What is neuronal polarity? | The establishment of neuronal asymmetry with morphologically and molecularly different counterparts |
| What guides the dorso-ventral guidance of the HSN neuron? | Unc-6 and Unc-40 |
| What happens to the HSN polarity when you KO unc-6 and unc-40 | Dorso-ventral assymetry is not generated |
| Where is Unc-40 localised on the HSN neuron? Is the action of Unc-40 reliant on unc-6 | Asymmetrically localised on ventral side (axon grows out from ventral side). Yes Unc-40 assymetry is lost in unc-6 mutants. Therefore, UNC-6/Netrin is a polarity molecule. It regulates HSN dorso-ventral neuronal polarity through the UNC-40/DCC receptor. |
| Which molecules regulate neuronal polarity on the anterior posterior axis? (c.elegans) | WNT molecules also function in neurone polarity on the anterior posterior axis |
| What C.elegans axon can be used to study the orientation of neuronal plasticity in the anterior posterior axis in vivo? Describe it | PLM .Has a long anterior process, with synapses and growth cones, A short posterior end with no synapses or growth cone |
| What molecule controls the anterior-posterior polarity of the PLM axon? What is its receptor? Do they work in the same genetic pathway? Are they autonomously required? Are they repellant or repulsive? | The WNT molecule lin-44. It's Frizzled receptor is Lin-17. They work in the same genetic pathway. They are autonomously required. Repulsive - localised to posterior side |
| How can synapses be visualed in vivo (C.elegans) | GFP or RFP can be tagged to a pre-synaptic protein (RAB-3) under control of specific promotor |
| How are DA9 motoneurons and its synapses visualied in C.elegans | RFB is tagged to RAB-3 pre-synaptic protein under control of the mig-13 promotor |
| What molecules are required to correctly position the DA9 synaptic assembly (C.elegans) | The Wnt molecule Lin-44 and and Frizzled receptor Lin-17. The mutants have extra synapses on the DA9 processes |
| Localization of LIN-17 defines an __ domain in Da9 neurona. | Asynaptic domain. Whenver LIN-17 is expressed, pre-synaptic loci cannot form. Therefore, Wnt signals and Frizzled receptors inhibit synapse formation at the neuromuscular junction. |
| Does LIn-17 function autonomously in DA9? | Expressed wild type Lin17 in DA9 neurons, in an animal which is mutant for LIN17It was sufficient to rescue, therefore autonomous |
| Is lin-17 localisation in DA9 LIn-44 dependent? | Yes, Lin-44 KO had lin-17 expressed ubiquitously in Da9 neurons |
| Does lin-17 act as in inctructive cue in DA9 neurons? | To determine whether Lin44 acts as a permissive or instructive cue, you ectopically express Lin44 and measure phenotype It was instructive as the asynaptic side moved |
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Lui24