Optogenetics

Question 1

Describe the visualisation of optogenetic manipulation using Ca2+ levels

Answer 1

• Calcium sensor GCaMP levels to read out neuronal activity
• Modification of GFP with addition of Ca2+ binding domain from another protein
• When Ca2+ to the domain conformational change within the protein which emits fluorescence hence enabling visual read out of neuronal activity

Question 2

Describe the experiment conducted on nucleus of medial longitudinal fasciculus in zebrafish to investigate its behavioural role. Detailed the optogenetic techniques used to conduct this experiment

Answer 2

• This experiment demonstrate the power of using optogenetics to elucidate the central function of nMLF
• First, they used calcium sensors at nMLF to show that nMLF cells are broadly and bilaterally activated during behaviour. This was done through quantitating Ca levels in cell
• Next, they used optogenetics (ChRh2) to activate nMLF which causes smooth tail deflections
• They also localised a stimulation to specifically pin point cells that are responsible for evoking the response including the large MeL neurons and MeS cells
• They showed that MeL and MeS contributed to steering behaviour by laser ablations which showed no movement

Question 3

Name three problems faced with using optogenetic approach

Answer 3

1. You have a risk of driving neuronal response outside the physiological range, might cause unnatural plasticity in the circuit
2. Not uniform optogenetic expression across target neuron - heterogeneity in magnitude of response
3. Implementation of optogenetic probes can perturb the system being investigated - high level and long-term expression of ChR2 has recently been shown to cause abnormal axonal morphology

Question 4

Describe the advantages and the potentials of optogenetics

Answer 4

1. Mimics natural pattern activity in neural populations in vivo by targeting multiple neurons for photoactivation and inactivation: precautions need to be made when targeting individual neurons in vivo to ensure reliable generation of individual spikes in a given neuron
2. It provides potential for all-optical approaches combining optogenetic expression and sensors to allow read out

Question 5

Name the methods that can be used to target specific subset of neuron to express the opsin gene and provide limitations where necessary

Answer 5

1. Viral expression: fast/ versatile, high infectivity BUT promoter fragment size limited to 4kn
2. Transgenic animals/ knock-in: costly and time consuming
3. Cre/low based system