Optogenetics

Question 1

What is the definition of optogenetics

Answer 1

• Combination of genetic and optical methods to achieve gain or loss of function of well-defined events in specific cells of living tissue

Question 2

Detail two types of control tools

Answer 2

Opsins:
- Type I opsins (microbial) and Type II opsins (animal)
- Both are GPCRs that require RETINAL, a vitamin-A related organic cofactor acting as an antenna for photons
- When retinal is bound, the functional opsin proteins are termed RHODOPSINS

Channelrhodopsins:
- Act as photo-receptors in unicellular green algae - control photo axis
- channelrhodopsin-1 and channelrhodopsin-2 from C. reinhardtii
- React to light by transporting ions across lipid membranes of cells in which they are genetically expressed

Question 3

What are 3 examples of natural rhodopsins and their qualities on murine neurons

Answer 3

Archaerhodopsins + Bacteriorhodopsins:
- 100% Neural silencing in cortical neurons of awake mice

Halorhodopsins:
- Quieted by yellow light

Channelrhodopsins:
- Respond to train of blue light

Question 4

What are 3 methods of producing cells that can be targeted using optogenetics (+Pros and Cons)

Answer 4

Viral expression
- Fast, versatile implementation, high infectivity for robust expression levels

Transgenic animals
- Cost +time intensive in rodent models

Cre/Lox systems
- Combination of Cre-dependent viral systems with existing cre driver lines

Question 5

How does optogenetics actually work?

Answer 5

• Project light onto the cortex, influencing neuronal firing
• Can take readouts of electrical activity / voltage indicators (QuasAr1/2)
• Computational analysis of imaging can give quantitative data
• Example; GCaMP is a fusion protein of GFP with a calcium binding protein - Calmodium + M13 - upon binding, conformational change leads to GFP fluorescing

Question 6

Outline the use of Zebrafish in the study of descending control of swim posture (Hausser et al 2014)

Answer 6

• Zebrafish are good as an be analysed high throughput and are transparent
• Hausser investigated behavioural role of the midbrain nucleus of the medial longitudinal fasciculus (nMLF)
• Thy expressed channelrhodopsin-2 in the nMLF
• Delivered optic fibre to the fishes brain
• Stimulation resulted in tail movement (gain of function expt)
• To identify which neurons were involved in tail movement, a photo-converted fluorescent marker could be used (track which neurons stimulated)

Question 7

What were the key findings of the Hausser et al study on zebrafish descending control of swim posture EXPT

Answer 7

• Using a combination of calcium imaging, optogenetic activation, and laser ablations, it was shown that a central function of the nMLF is to provide postural positioning of the tail during forward swims.
  
  • This happens likely via activation of the posterior hypaxial musculature.
  • This is primarily mediated by MeS (small medial) neurons and MeL (large medial) cells

Question 8

What are the main issues with optogenetic approaches?

Answer 8

• Bulk illumination results in heterogeneity of light exposure across target neurons (not uniform)
• The level of stim risks driving responses outside of physiological range causing unnatural plasticity
• Implementation of high levels of optogenetic probes can perturb the system, for example cause abnormal axonal morphology (seen with CHANNELRHODOPSIN-2)

Question 9

What does the future of optogenetics look like?

Answer 9

• Ability to mimic natural patterns of activity in neural populations in vivo (requires careful titration of light)
• Tech development to target deeper brain regions
• All-optical approaches combining expression of actuators, sensors, and readouts and control of the same neurons