Fragenkatalog Flashcards
What is Vectashield? (Inga)
- medium for mounting
- to adjust diffraction index
- prevents dehydration and bleaching of the sample
What is Triton and what is it used for? (Inga)
- Permeability: Triton makes pores in the membrane layer
- > it allows antibodies to enter the cell
Why use PFA (paraformaldehyde)?
- Fixation of proteins, carbons and lipids in the cell
- Cells survive further treatments
- stops all degradation processes
Why use PBS (phosphate buffer saline)? PBT?
- Wash the cells to remove fixation solution and unbound antibodies. PBT is PBS with Triton.
- For the washing and simultaneous creation of permeability
Why use NGS (normal goat serum)?
- Blocking to prevent / prevent non-specific binding of antibodies
- Non-specific epitopes are blocked to minimize that the second antibody binds them
Describe antibody staining. Why 2 antibodies?
- First antibody specifically binds to protein / structure to be labeled
- Second antibody specifically binds to the first antibody and is labeled with marker, e.g. Fluorophore, provided
- Cheaper to model specificity easier:
For frequently used structures there are already marker-causing first antibodies:
Nc82, N488: BRP
GluRII D, Cy3: glutamate receptor
Cy5: HRP (all neuronal membranes)
How does STED microscopy work?
Stimulated emission depletion
Fluorescence microscope, which also has a STED laser. This additional laser has a laser beam in the form of a donut (generated by a PhaseMod). In the middle of which the light with the exciting laser is located. This stimulates the photonons he meets. The surrounding photons are also excited, but depleted by the STED laser and its corresponding wavelength back to their original energy level, so that only the excited photons are emitted and detected. Resolution up to about 50 nm.
Rebecca: Stimulated emission depletion
Fluorescence microscope, equipped with two lasers: one for excitation and one for depletion (STED laser). A PhaseMod forms the light of the STED laser like a donut. This overlaps with the excitation laser. Fluophores are being excited everywhere where the excitation laser reaches but only the active focal spot emits photons, as the STED laser depleats the surrounding fluorescence. To do this, the STED laser is set to a specific wavelength, longer than the fluorescence, that stimulates red shifted emission of photons, which are not recognized by the detector.
Resolution up to about 50 nm.
What is fluorescence?
Spontaneous emission of light, shortly after excitation of a molecule (fluorophore). The emitted light is usually lower in energy than the previously absorbed.
Fluorophores are molecules that can be excited by light and emit longer-wavelength light.
Rebecca: The red shift (Stokes shift) in emitting light is caused by loss of vibrational energie in the excited state
What is a synapse? Which components are there (function)?
Is a transmission relay (chemical synapse (NMJ), electrical synapse with gap junctions and connetones).
Chemical synapse:
- Presynapse: with AZ, vesicles are collected on scaffold structure (BRP RIM binding protein, Syd-1 (Liprin, neurexin anchored)) and driven by conformational changes, by action potentials, to membrane fusion and thus to release
- Synaptic cleft: Neurexin and Neuroligin bind to each other. Vesicle diffusion to receptors at postsynapse
- Postsynapse: glutamate receptors GLRII A + B (calcium-dependent), neurologist. Docking and fusion of vesicles, signal transmission. In NMJ contraction of muscle cells.
What is a balancer? Function?
Chromosome provided with a dominant marker gene that does not allow (hardly) crossing-over (by inversions in the chromosome). Mutations in the cane can be achieved with this balancer because animals homozygous with the balancer chromosome are lethal.
• multiple inverted chromosome
• recessive lethal
• dominantly marked
Rebecca:Different balancers exist for 1st, 2nd and 3rd chromosome, each with a phenotypic marker. Homozygotes for balancers are lethal, also recombination of balancers leads to death just like homozygotes for the mutation.
Heterozygotes with balancer and mutation keep the line viable and also ensure the mutation in every fly
Advantages and disadvantages of Drosophila
Advantage: - small genome - known genome - low maintenance costs - saves space - relatively easy to manipulate - short life cycle disadvantage - Research results not transferable to humans - high risk of contamination - Materials can not be frozen, such as in bacteria - Difficult handling due to small size
What are micro-RNAi and their function?
- regulatory function, gene silencing
- Degradation of already synthesized mRNA
- Endogenously coding
- short, noncoding RNA
- at the post-transcriptional level
- Si: are not endogenous coding
- targeted RNA degradation
What is CRISPR and how does it work? Advantage disadvantage?
- search homologous sequences for guide RNA (s) (many Gs and Cs) by known sequence
- guide RNA (s) find homologous sequences through PAM structures
- homologous arms are designed
- Cas9 endonuclease / nickase cuts genomic target DNA
- Double-strand breakage may combine by homologous combination with cassette to insert or non-homologous recombinations may complete
Rebecca: The Crispr/Cas system consists of Crispr-RNA (cr-RNA), Cas9-Enzym and PAM sequence. Repeat sequences of cr-RNA bind to the Cas9 Enzyme, the guide RNA recognizes and binds specific DNA sequences upstream of PAM (NGG/NCC). Cas9 then creates a double strand break in the genome 3 base pairs upstream of PAM. DSB is repared, but not correctly. This leads to deletions or insertions ( knock outs). Homology directed repair with a homologous template leads to a knock-in or fusion.
Cas9 Nickase creates single strand breaks and minimizes off target effects. Using paired nickases creates two single strand breaks at different points. The resulting sticky ends make homologous recombination easier
Which memory phases are there in Drosophila?
Short term memory: for a few minutes, does not require repetitive training, in experiment flies are tested 5 min after exposure to stimuli
Mid term memory: for up to few hours; in experiment flies are tested 1h after exposure to stimuli or 3h if interested in ARM&ASM
Long term memory: for 24h up to a few days, requires repetitive training (e.g. 10 times training session); in experiments flies are tested 24h after training.
Anasthesia resistent/sensory memory: 2,5h after exposure to stimuli, flies receive a 90s cold shock followed by 30 min recovery time
-> ARM: ability to keep memory after shock
-> ASM= MTM – ARM
Why are 2 groups doing the short-memory test?
- olfactory learning
- Eliminate errors in fragrance dosing
- Results must be averaged to rule out that the flies from the outset prefer a particular fragrance -> normalization to fragrance preference
Odor preference is always there, stronger in one direction as Shock and Odor preference come together. Data fusion balances preference
What is a neuropil and which neuropil in insects is known as a center for learning and memory? Describe the structure.
Neuropil: Synaptic tissue in which few to no cell bodies are present
Mushroom body (alpha, ß, alpha’-, ß’- and gamma-neurons): from different lobes
Formed from Kanyon cells, it is innervated from projection neurons in the calyx
What is a NMJ and which function does it have?
- Structure between nerve and muscle cells
- Serves the innervation of the muscle by motor neurons
- Control of muscle contraction
- Synaptic signal transmission
- Regulation of the Ca2 + influx at the postsynapse
- contains many synapses
What is Bouton?
Axonswelling to create synapse connection (many synapses)
What is an active zone (AZ)? Which structure, function? Main components?
Place where the synaptic vesicles fuse with the membrane. Scaffold is the scaffold of the AZ, which stabilizes the AZ and serves to facilitate vesicle fusion. Proteins for building the scaffold -> dustering of Ca channels
- The membrane area at which the vesicles fuse
- AZ scaffold, are the scaffold proteins responsible for the release
- Ca2 + channels are clustered there
- ELKS / CAST family
~ BRP
~ RIM-BP
~ UNC13
~ Liprin-a
~ Syd-1
~ (Piccolo and Bassoon)(mouse)
What is GCamp, what does it feel, function?
- Calcium indicator
- Consists of Calmudulin, M-13 and GFP
- changed conformation change
- viewable in vivo
- postsynaptic
What is the normal resting potential in mV for muscle 6?
-60 to - 70mV
What is a mEJC or miniature excitatory junctional current?
An mEJC is a postsynaptic response to a spontaneous presynaptic release from a vesicle
Paired-plus experiment: what is measured and what can one conclude from it?
Experiment in which two consecutive stimuli are delivered in one at a short inter-stimulus interval (10 or 30 ms in our case). This type of experiment is used to gain insights into short-term synaptic plasticity and the likelihood of vesicle release.
What is the function of the three electrodes used in the TEVC (two electrode voltage clamp)?
Two pointed microelectrodes are attached during normal recordings, a microelectrode serves for constant recording of the membrane potential (command potential). In the third electrode, the motor neuron is introduced, which innervates the desired muscle by a stimulus.
What is the most important cation for proper synaptic transmission in Drosophila NMJ? What is the physiological extracellular concentration of this cation?
Ca2 + is the most important cation for synaptic transmission to Drosophila NMJ. During TEVC recordings, CaCl2 is added to the bath solution to achieve the approximate physiological extracellular concentration of 1-1.5mM.
What is a neurotransmitter in Drosophila NMJ?
glutamate
How would one determine the possible number of SVs distributed after a single action potential?
First, measure the amplitude of the postsynaptic response to a single vesicle (mEJC). Use simulation protocol to determine the evoke excitatory junctional current (eEJC) amplitude (response to many vesicles followed by an action potrntial) eEJC can be divided by average mEJC amplitude to determine the approximate number of vesicles that were released following a single action potential.
What are components of a good experiment?
- Hypothesis: statement that should be checked for its truth content
- Controls (+/-)
- Objectivity (different experimenters = result)
- Reability (reproducibility (biological, technical replicates))
- Validity (validity, exclusion of disturbance variables)
- dependent variables
Which lines did we use?
Brain: Orco-Gal4 (III) x UAS: mcD8-GFP (III) GHI46-Gal4 (III) x UAS: mcD8-GFP (III) MB247-Gal4 (III) x UAS: mcD8-GFP (III) NMJ: Group A: Ok6-Gal4, UAS: dicer2 x UAS: BRP-RNAi (B3/C8)(III) Ok6-Gal4, UAS: dicer2 x W1118 (PFA Fixation) Group B: Cac::sfGFP/X (Methanol Fixation) ERG: GMR-Gal4(X) x UAS: BRP-RNAi (B3/C8) (III) GMR-Gal4(X) x W1118
What is Gal4-UAS doing?
To study gene expression and function of organisms.
Using the Gal4-UAS system, specific cloned genes can be used to express specific cells / cell types or tissues.
Gal4 is a helper-specific transcriptional factor (transcriptional activator) under the control of a weak promoter -> encodes Gal4, enhancers are needed.
Protein Gal4 binds specifically to the upstream activating sequence (UAS), which activates a downstream target gene
System comes from yeast.
- > localisation: constitutive Gal4 system
- > time point: inductible Gal4 system
What is an enhancer?
An enhancer is a promoter enhancer. The promoter would not be active without the enhancer or would work very poorly
