Visualisation and identification of neurons and networks - Beed

Question 1

3 ways to slice the brain

Answer 1

• Coronal
• Horizontal
• Sagital

Question 2

what are the 2 types of visualisation?

Answer 2

1. Static visualisation:
   - good for cell counting
   - done on fixed network
   - can be done on cell (Nissl staining/DAPI), mRNA (ISH) or protein (IHC) level
2. Dynamic visualisation:
   - measurement and identification of neuronal responses w. detailed morphology (input/output)
   - provides better understanding of neuronal communication in networks
   - done on living tissue

Question 3

what was the first (modern?) neuroimaging technique?

Answer 3

labelling cells with GFP and imaging with fluorescence microscopy

Question 4

What is DIC microscopy?

Answer 4

Differential Interference Contrast microscopy;

• an optical microscopy technique
• used to enhance the contrast in unstained transparent samples
• based on the concept of interferometry to gain info about optical path length of the sample and thus increase the resolution

Question 5

how can single neurons/tissues be labelled using patch clamp?

Answer 5

• patch clamp is a technique used to recored electrical activity of neurons and tissue by injecting current to induce AP
• -> at the same time, it is possible to inject different substances to “fill” the cell in order to distinguish between different parts (morphology) of neurons and look at what parts of the cell are active
• substances injected to the cell are chosen according to the type of connectivity of interest (e.g. functional/structural)
• commonly used substances: biocytin, fluorescent dyes

Question 6

what are the differences between fluorescent dyed and biocytin as cell markers? (when to use them)

Answer 6

Biocytin: is used to look at functional connectivity. shows how morphology and firing patterns together identify the cell. however, visualisation is possible only post-experiment)

Fluorescent dye: used to look as structural connectivity. shows morphology and firing pattern by adding colours according to change in fluorescence of the structure, but no temporal function can be visualised (only live morphology).

Question 7

how does Ca2+ dye work (for visualisation)

Answer 7

• Ca2+ is used to measure chemical activity in neurons
• makes it possible to look at activity in dendritic spines
• how does it work?- Ca2+ dye is a fluorescent dye that changes its fluorescence with increase in calcium concentration
• alternative: voltage sensitive dye to measure electrical activity and allow a fast AP induction
• source of Ca2+- AMPAR and NMDAR let Ca2+ into the spine

Question 8

Advantages of Ca2+ dye and voltage sensitive dye

Answer 8

• do not require patching the neurons because the structures and the substrate for the dyes naturally occurs in the cell
• makes it possible to study many things both in-vivo and in-vitro

Question 9

a. What are opsins?

b. Name 3 different types

Answer 9

a. - opsins are light sensitive channels, found in algae and bacteria, which can be introduced to a neuron and activate it when light is flashed on the neuron.
- following activation, they can lead to depolarisation of the cell (enhancers) or hyperpolarisation (silencer)
- they are the base of optogenetic.

b. Types:
- Channelrhodopson- pumps Na+, Ca2+, K+ and H+ into the cell -depol
- Halorhodopsin- Cl- channel inserted into neuron–> hyperpol.
- Bacteriorhodopsin- pumps protons out of the cell - hyperpol.

Question 10

Can different opsins (e.g. channelrhodopsin and halorhodopsin) be activated in the same cell? how?

Answer 10

Yes. both can be expressed and activated in the same cell, since they have different activation spectra. (activating them requires lights with different wavelengths)

Question 11

structure of channel-/halorhodopsin

Answer 11

• normally exist in an inactive form (trans)
• activation upon light–> change of conformation from trans to cis
• advantage: reversibility- the structure and change of conformation depends on light. I.e. light goes off–> channel closes and goes back to its trans, inactive form

Question 12

activation spectra of channel-/halorhodopsin

Answer 12

channelrhodopsin- 325-600 nm (peak at 425 nm)

halorhodopsin- 450-725 nm (peak at 625 nm)

Question 13

ways to express opsins in neurons

Answer 13

1. non-specific expression- artificial channel induced in virus–> infecting the brain w. the virus–> channel expression in all neurons
2. specific expression- are-dependent AAV- are factors (driver) recognise specific gene sites –> are is induced w. channelrhodopsin and introduced to neurons –> are binds only to specific cells that recognise it –> neuron specific expression

Question 14

How can we see (image) optogemetically modified neurons?

Answer 14

tagging the rhodopsin with a fluorescent protein (e.g. GFP) which has a different wavelength from the rhodopsin activation spectrum.

Question 15

why is it important to choose a fluorescent protein with an activation wavelength different from the one of rhodopsin?

Answer 15

activation ≠ visualisation
if activation and visualisation wavelengths are the same –> the wavelengths will interfere with each other.

Example:
ChR- active in blue light; vis. in yellow light
HR- active in orange light; vis. in green light