Techniques and tools for studying synapses Flashcards
What are the different experimental approached for studying synapses
electrophysiology, electron microscopy, fluorescence imaging ( FM dyes, calcium imaging, genetically encoded indicators), Molecular biology, and tools for manipulating synaptic activity.
Describe Bernard and qunatal transmitter relase
He found using diff concentration of external Ca2+ in saline makes end plate potentials differ in amplitude.
what were the three conclusions made by Bernard Katz
- End plate potentials consist of multiple, mini-like quanta of neurotransmitters.
what were Bernard Katz’s hypothesis based on
electrophysiological recordings
With a higher Ca concentration we see a larger ______________ _____________
evoked response
Explain extracellular tracers and entry into synaptic vesicles following stimulation
we use extracellular tracers to show this idea that synaptic vesicles can fuse with the plasma membrane that opens and in this case allow for tracers to be taken up.
What is electron microscopy commonly used for?
Used to look for changes in the number of synaptic vesicles and active zone since they are the site of neurotransmitter release. We can also look at the number of synaptic vesicle pools and the number of synaptic vesicles.
Fluorescence imaging (FM dyes)
FM1-43 and FM4-64 are lipophilic styryl dyes
Measuring synaptic vesicle cycling using FM dyes.
• we can apply the FM dye to the saline but without stimulation, the dye will remain external to the presynaptic terminal
Described how FM dyes work, and explain how they are used to measure synaptic vesicle cycling, exocytosis and endocytosis?
Exocytosis: FM dyes are used to visualize exocytosis. As synaptic vesicles fuse with the plasma membrane and release neurotransmitters into the synaptic cleft the FM dye molecules are also expelled into extracellular spaces. This results in a increase in fluorescence intensity. The rate and extent of dye release correlate with the rate and extent of synaptic vesicle exocytosis.
Fluorescence imaging (calcium imaging) What are the different types of calcium imaging? describe them breifly
A. Single-cell loading techniques
Fluorescense ________________when stimulated and _______________when not stimulated
Increases, Decreases
Fluorescence imaging (genetically encoded indicators) Describe genetically encoded calcium indicators
GcAMP can be expressed in the pre or post synaptic neuron.
Overtime the Ca indicators have gotten __________
better and better
Fluorescence imaging (genetically encoded indicators) describe genetically encoded transmitter indicators
• Genetically encoded transmitter measures changes in glutamate
Fluorescence imaging (genetically encoded indicators) Describe synaptopHluorin
• pH-sensitive GFP molecule is fused to synaptopHluorin and it is in the lumen of the vesicle
Describe syantopHluorin and how they work and explain how it is used to measure synaptic vesicle cycling, exocytosis and endocytosis
• SyanotoPHorin molecule is expressed in synaptic vesicles, where there is a low fluorescence intensity.
___________indicators can be used to measure changes in membrane potential
Voltage
Name 2 molecular biology techniques
- Changes in mRNA or protein expression
what are 6 tools to target your gene or protein of interest
- Knockout or null mutants
western blotting is used to measure ___________
proteinn expression
qRT-PCR is used to measure
mRNA expression
Co-immunoprecipitation is used to
determine if two protein bind together
Describe geentic engineering
• Tools used to create a loss of functions or overexpression
describe knocouts or nulls
The protein of the gene affected is not expressed. This can be due to a mutation, partial deletion, or complete deletion of the gene.
describe hypomorph mutant
A mutation causes a partial loss of gene function through reduced protein expression.
What is the difference between a hypomorph mutant and a null or knockout
Hypomorphism causes partial loss of gene while null or knockout causes a complete loss of gene.
Describe RNAi
RNAi occurs when double-stranded RNA activates an enzyme called dicer which cleaves double-stranded RNA ar staggered positions creating siRNA
what are some off target effects when dealing with RNAi
There are some differences in dendritic structures and spine densities
dominant negative approches
• An altered gene product that acts antagonistically to the wild-type allele
Overexpression
• Increasing the exression of your protein of interest
Fluorescein assisted light activation
• based on the use of an epitope tag on the protein of interest
Tools for munipulating synptic activity (Shibire)
• Encodes a protein called dynamin that is essential for endocytosis
Tools for munipulating synptic activity (Dynasore)
Cell permeable inhibitor of dynamin and acts as a protein inhibitor of endocytic pathways known to depend on dynamin.
Tools for munipulating synptic activity (Botulinum Neurotoxins)
• Botulinum neurotoxins can cleave snare proteins disrupting their complex and blocking synaptic vesicle fusion and endocytosis
Tools for munipulating synptic activity (Optogenetics)
Involves the use of light to acutely control the activity of the neuron
Tools for munipulating synptic activity (Channelrhodopsin)
Channelrhodopsin are light-gated ion channels that enable single-celled motile algae to seek ambient light conditions suitable for photosynthesis an survival
