Techniques and tools for studying synapses

Question 1

What are the different experimental approached for studying synapses

Answer 1

electrophysiology, electron microscopy, fluorescence imaging ( FM dyes, calcium imaging, genetically encoded indicators), Molecular biology, and tools for manipulating synaptic activity.

Question 2

Describe Bernard and qunatal transmitter relase

Answer 2

He found using diff concentration of external Ca2+ in saline makes end plate potentials differ in amplitude.

Question 3

what were the three conclusions made by Bernard Katz

Answer 3

1. End plate potentials consist of multiple, mini-like quanta of neurotransmitters.

Question 4

what were Bernard Katz’s hypothesis based on

Answer 4

electrophysiological recordings

Question 5

With a higher Ca concentration we see a larger ______________ _____________

Answer 5

evoked response

Question 6

Explain extracellular tracers and entry into synaptic vesicles following stimulation

Answer 6

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.

Question 7

What is electron microscopy commonly used for?

Answer 7

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.

Question 8

Fluorescence imaging (FM dyes)

Answer 8

FM1-43 and FM4-64 are lipophilic styryl dyes

Question 9

Measuring synaptic vesicle cycling using FM dyes.

Answer 9

• we can apply the FM dye to the saline but without stimulation, the dye will remain external to the presynaptic terminal

Question 10

Described how FM dyes work, and explain how they are used to measure synaptic vesicle cycling, exocytosis and endocytosis?

Answer 10

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.

Question 11

Fluorescence imaging (calcium imaging) What are the different types of calcium imaging? describe them breifly

Answer 11

A. Single-cell loading techniques

Question 12

Fluorescense ________________when stimulated and _______________when not stimulated

Answer 12

Increases, Decreases

Question 13

Fluorescence imaging (genetically encoded indicators) Describe genetically encoded calcium indicators

Answer 13

GcAMP can be expressed in the pre or post synaptic neuron.

Question 14

Overtime the Ca indicators have gotten __________

Answer 14

better and better

Question 15

Fluorescence imaging (genetically encoded indicators) describe genetically encoded transmitter indicators

Answer 15

• Genetically encoded transmitter measures changes in glutamate

Question 16

Fluorescence imaging (genetically encoded indicators) Describe synaptopHluorin

Answer 16

• pH-sensitive GFP molecule is fused to synaptopHluorin and it is in the lumen of the vesicle

Question 17

Describe syantopHluorin and how they work and explain how it is used to measure synaptic vesicle cycling, exocytosis and endocytosis

Answer 17

• SyanotoPHorin molecule is expressed in synaptic vesicles, where there is a low fluorescence intensity.

Question 18

___________indicators can be used to measure changes in membrane potential

Answer 18

Voltage

Question 19

Name 2 molecular biology techniques

Answer 19

1. Changes in mRNA or protein expression

Question 20

what are 6 tools to target your gene or protein of interest

Answer 20

1. Knockout or null mutants

Question 21

western blotting is used to measure ___________

Answer 21

proteinn expression

Question 22

qRT-PCR is used to measure

Answer 22

mRNA expression

Question 23

Co-immunoprecipitation is used to

Answer 23

determine if two protein bind together

Question 24

Describe geentic engineering

Answer 24

• Tools used to create a loss of functions or overexpression

Question 25

describe knocouts or nulls

Answer 25

The protein of the gene affected is not expressed. This can be due to a mutation, partial deletion, or complete deletion of the gene.

Question 26

describe hypomorph mutant

Answer 26

A mutation causes a partial loss of gene function through reduced protein expression.

Question 27

What is the difference between a hypomorph mutant and a null or knockout

Answer 27

Hypomorphism causes partial loss of gene while null or knockout causes a complete loss of gene.

Question 28

Describe RNAi

Answer 28

RNAi occurs when double-stranded RNA activates an enzyme called dicer which cleaves double-stranded RNA ar staggered positions creating siRNA

Question 29

what are some off target effects when dealing with RNAi

Answer 29

There are some differences in dendritic structures and spine densities

Question 30

dominant negative approches

Answer 30

• An altered gene product that acts antagonistically to the wild-type allele

Question 31

Overexpression

Answer 31

• Increasing the exression of your protein of interest

Question 32

Fluorescein assisted light activation

Answer 32

• based on the use of an epitope tag on the protein of interest

Question 33

Tools for munipulating synptic activity (Shibire)

Answer 33

• Encodes a protein called dynamin that is essential for endocytosis

Question 34

Tools for munipulating synptic activity (Dynasore)

Answer 34

Cell permeable inhibitor of dynamin and acts as a protein inhibitor of endocytic pathways known to depend on dynamin.

Question 35

Tools for munipulating synptic activity (Botulinum Neurotoxins)

Answer 35

• Botulinum neurotoxins can cleave snare proteins disrupting their complex and blocking synaptic vesicle fusion and endocytosis

Question 36

Tools for munipulating synptic activity (Optogenetics)

Answer 36

Involves the use of light to acutely control the activity of the neuron

Question 37

Tools for munipulating synptic activity (Channelrhodopsin)

Answer 37

Channelrhodopsin are light-gated ion channels that enable single-celled motile algae to seek ambient light conditions suitable for photosynthesis an survival