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Neural Circuits > L3 - Methods in Modern Neuroscience > Flashcards

L3 - Methods in Modern Neuroscience Flashcards

1
Q

What are the 4 methodological approaches to study new neuronal types?

A

Describe morphology – where projections are sent, size of dendritic tree
Map connections – which cells it connects with
Describe activity – which stimuli activate neurons
Theoretical study

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2
Q

Morphological studies - Cajal and Golgi

A

Development a method to label cells – don’t label every cell

Allowed them to characterise the morphology of the neurons

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3
Q

Sparse labelling was discovered by?

A

Golgi

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4
Q

Fine details were discovered by?

A

Cajal

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5
Q

What are the problems with staining morphology?

A

Does not allow for staining of individual neurons
Cannot combine electrophysiology and morphology assessment of same cell
- Staining kills the neuron

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6
Q

What was the solution to the problems with staining morphology?

A

Patch clamp

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7
Q

Patch clamp method - electrophysiology

A
  1. Use glass electrode filled with electrolyte - similar to intra- or extracellular solution
  2. Touch the cell and apply negative pressure
  3. Part of the membrane will form tight junction with the membrane
  4. Can record currents through this patch
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8
Q

How can you record currents through the whole cell?

A

Can break the membrane

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9
Q

How do you carry out morphology and electrophysiology in one?

A

Adding fluorescent dye in the pipette solution will give information about the morphology of recorded neuron

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10
Q

What are the problems with electrophysiology and morphology?

A

Cannot label many cells
Limited ability to label specific cell type
Limited ability to label cellular compartments
Limited ability for live labelling- mostly uses slices of tissue

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11
Q

What is the solution to the problems with electrophysiology and morphology?

A

GFP

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12
Q

What is GFP?

A

Fluorescent protein
Stimulated by blue light
Emits green light

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13
Q

What can GFP be used for?

A

Used to understand morphology and neuron function

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14
Q

What are the differences between fluorescent proteins?

A

Can have different excitation and emission wavelengths

All have different colours

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15
Q

How does a

simple fluorescent microscope work?

A

Need an excitation light – use a dichroic mirror

  • Reflects light of just one wavelength
  • Every other wavelength passes though – light we see
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16
Q

What is GCaMP?

A

GFP based calcium indicator

17
Q

What is the structure of GCaMP?

A

Fusion of GFP and two calcium binding proteins

- M13 and Calmodulin

18
Q

How does GCaMP work?

A

In presence of calcium the two proteins interact
GFP becomes much brighter
Used to understand the function of neurons

19
Q

What is GCaMP role in active neurons?

A

Expressing GCaMP means can observe the activity in the neurons
When neuron is active lots of Ca channels on the membrane opens and Ca in cytoplasm rises
Neurons then become brighter
Can image the entire brain with a single neuron resolution

20
Q

What are the two types of high resolution microscopes?

A

Wide field microscopy
Confocal microscopy
Allows for massive increase in spatial resolution

21
Q

Wide field microscopy

A

Collects light from, below and above the focal plane

22
Q

Confocal microscopy

A

Rejects light coming not from the focal plane
Decreases the area of excitation
Higher resolution

23
Q

What are the problems with in-vivo methods?

A

Animal is sedated using Na+ channel blockers
Animal is stressed
Animal does not perform behaviour that it usually does

24
Q

What are the solutions to the problems with in-vivo methods?

A

Virtual reality

Freely moving animal

25
Q

Virtual reality with mouse

A

Mouse is placed on a ball that moves in certain direction when mouse moves
Turn of the ball leads to turn of the field of view
Head is still fixed
Objective is placed on top as usual

26
Q

Free moving mice

A

Tiny fluorescent microscope placed directly in the skull

Image delivered via light guide

27
Q

Neuronal stimulation by light - channelrhodopsin

A

Non-specific ion channel activated by light
When channel opens, cell depolarizes
Activated by blue light – channel opens

28
Q

What is the problem with blockers?

A

They are not very specific – diffuse

29
Q

Hyperpolarisation by light - halorhodopsin

A

Specific for chloride ions
Activated by yellow light – channel opens
When channel opens, cell is hyperpolarized

Neural Circuits (15 decks)

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