L5 - Optical approaches to studying circuits

L5 - Optical approaches to studying circuits

1. 1. TYPES OF APPROACHES

   • a. Simple systems
     • Aplysia feeding network has had many properties defined
       • Synaptic properties
       • Wiring properties
       • Endogenous properties
       • Allows us to build up a network to understand the feeding system
     • C. elegans
       • Entire neural network has been identified
         • 302 individual neurons
           • Categorized into 118 groups
         • 7 pharyngeal muscles
           • 1 muscle class
   • c. Complex systems
     • Individual neurons within complex circuits can be complicated due to number of dendritic trees
       • Neurons are not uniquely identified - homogenous populations are used
     • Difficult to study
       • Brain slices require patch methods
       • Chances of choosing two liked neurons under a microscope is low
       • Using neurobiotin on neurons to turn them black enables microscopy methods to reconstruct a 3D representation
   • b. Microelectrode arrays
     • If the only information required is overall spike activity, then this method is sufficient
       • No info on neural wiring
       • No info on neurophysiology
     • Nanoscale plates with multiple electrodes that can pick up local voltage changes
       • Non-specific - takes random samples rather than a specific neuron
     • Do NOT detect sub threshold activity, only APs
     • Works best in cultured neurons
   • d. Optical approaches
     • Light avoids the need for physical contact with the tissue by travelling through membranes
       • An electrode will tear the membrane, this could impact biophysical properties
     • Provides high spatial resolution
       • By recording from multiple locations at the same time
     • Genetic tools allows optical probes to target

2. 2. BRAINBOW

   • a. Function
     • Modern multi-colour Golgi stain
     • A cDNA construct based on Cre-Lox recombination, it codes for 4 different fluorescent proteins
       • GFP - Green Fluorescent Protein
       • Red Fluorescent Protein
       • Yellow Fluorescent Protein
       • Cyan Fluorescent Protein
       • Under normal conditions, only GFP is transcribed
         • This is related to loxP promoter sites
         • Adding Cre recombinase enzyme produces four possible outcomes leading to the four protein types
         • Using multiple copies of brainbow produces a wider variety of colours
     • Non-selective - random
   • b. Use in mapping and tracing
     • Can track anatomy of populations through tissue
     • Doesn't provide info on connectivity/how neurons are wired together

3. 3. VIRAL TRACING

   • a. Method
     • Rabies virus used
     • Virus can infect a neuron and cross its synapses in a retrograde direction (post to pre)
     • i. Coating of the virus is removed i.e. genes encoding the coat are removed
       • ii. A GFP is added to track where the virus is expressed
         • iii. The missing gene for the coat is kept as a separate plasmid
           • iv. A target neuron is infected by injecting the virus into the cell body as well as the plasmid containing the coating gene
             • v. The virus lacks the gene require to allow synapse crossing, the plasmid solves this problem via transcomplementation. Alllowing retrograde movement backwards.
               • vi. However, as the virus does not contain the gene, it is limited to going back one step only. This prevents continuous injection, which would light up the whole brain.
     • Targeting can be tested and proven using electrophysiology and patching

4. 4. MANIPULATOR: OPTOGENETICS (channelrhodopsin - ChR2)

   • Manipulator: used to interrogate the system, modulate some aspects of neuromodulation and see how the system responds
   • i. Discovery
     • i. Is a protein isolated from algae, where phototaxic responses occurred, with sensitivity in the eye area
       • ii. Scientists purified this and inserted it into a frog egg (as they do not express many proteins themselves)
         • iii. Once the proteins from the eye spot were expressed, the egg became light sensitive
           • iv. By shining light onto it, changes in electrical properties occurred (observed using a voltage clamp)
   • ii. Function
     • Sequencing found it is a common ion channel used for natural light sensitivity
       • With 7 transmembrane domains and a central pore
         • Contains retinal - absorbs photons to cause conformational change in ion channels
           • This causes the pore to open and allow cations to move in
             • No selective - most positive cations can pass through
             • Leading to depolarization
   • iii. Uses
     • Able to control neural firing using light
       • Genetic modification of ChR2 using kinetics, spectral sensitive and trafficking
       • Targeting cDNA of promoters upstream of ChR2
       • Transgenic mice allowed light to switch neurons on and off, this can be used to drive motor activity of tested animals
       • Tested the direct and indirect pathways of motor planning in the basal ganglia (direct = facilitates movement, indirect = inhibits movement)
         • ChR2 optogenetics tested the pathways independently
           • Excitation of the indirect pathway caused 'parkinsonian state'
             • Mouse model of P disease, direct pathway activation rescued deficits in bradykinesia. Potential therapeutic strategy?
           • Bilateral excitation of direct pathway increased locomotion
   • iv. Halorhodopsin
     • Light driven chloride channel, NpHR
     • Ch2R and NpHR can control excitation and inhibition
       • Synthetic molecular photoswitches bind directly to ion channels to make them light sensitive - therapeutic value?
     • Hyperpolarizes neurons

5. 5. PASSIVE REPORTER: VOLTAGE SENSITIVE DYE

   • b. New reporters
     • GluSnFR
       • Glutamate sensor
       • When placed in the synaptic cleft, it responds to levels of glutamate
     • GCaMP and SyGBaMP
       • Genetically encoded reporters of calcium that allow targeting of specific subtypes of neuron
         • This allows targeting of specific subtypes of neuron
   • Passive reporter: provides simple readout properties of the neuron that were occurring anyway
   • a. Background
     • Dyes that alter their spectral chracteristics in response to voltage changes
     • Provide a total solution to visualizing neural circuit activity
     • CONS
       • Poor signal to noise
       • Diifcult to gain single cell solution