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Lecture 9

Lecture 9.docx

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University of Toronto Scarborough
Rutsuko Ito

Lecture 9 Light activated brain - Optogenetics Lecture outline 1. Focus on page 1-22 on optogenetic review handout Challenge in neuroscience 1. Challenge of heterogeneity of neurons in the brain 2. Existing in vivo techniques (electrophysiological, pharmacological, lesion) do not have spatiotemporal precision to be able to distinguish different cell types 3. Need to control one type of neuron in the brain, while leaving others intact 4. Francis Crick a. Speculated that light may have properties to serve as a neural control tool Method of the year 2010 Optogenetics a. The combination of optic and genetic techniques to control the activity of individual neurons on a millisecond scale Cellular mechanisms of driving neuronal activity with light Discovery of microbial opsins (Late for class, missed everything up to this point) 1. Bacteriorhodopsin (BR) a. Single component (type I) proton pump that is activated by photons of green light 2. Halorhodopsin (HR) a. Halobacteria chloride ion channel pump activated by green / yellow light 3. Channelrhodopsin (ChR) a. Single component cation channel expressed in green algae (chlamydomonas reinhardtii) activated by blue light to cause depolarization Using light to control cellular activity 1. Phototransduction in retina a. Rhodopsin i. Mammalian (type II) opsin in which the light sensing and ion conductance are carried out by different proteins 2. Bacterial rhodopsin vs mammalian rhodopsin a. Analogous to ionotropic vs metabotropic receptors i. Type I  ionotropic (fast, singular) ii. Type II  metabotropic (slop, bi-parted) Optogenetic tool variants 1. Microbial opsins are used as optogenetic tools 2. Further molecular engineering (e.g. point mutation) of single component microbial opsins can generate a whole plethora of variants with different kinetic and spectral properties 3. Example exam question: a. Which type of opsin would be preferably used according to graph on slide 12? b. No need to memorize graph (will be given) Optogenetic tools for excitation 1. Demonstrating that a neural population is causally sufficient for a behavioral property a. For higher expression levels of opsin genes for in vivo work i. Algal codons are replaced by mammalian codons  codon optimization (humanized ChR2) 2. Point mutations need to be optimized for desired physiological effect a. H134R mutation in ChR2 increases photocurrent magnitude by 2 fold, but causes 2 fold slower channel closure kinetics i. Great for inducing low frequency spike trains b. ChETAs (ChR E123T/A) generated by modifying ChR2 residue glutamate 123 to threonine / alanine i. Accelerates channel closer kinetics from ~10 to ~4 ms 1. Albeit at a cost of reduced photo current magnitude 2. Great in fast spiking cells Optogenetic tools for inhibition (didn't pay attention to this slide) 1. Excitation vs inhibition? 2. Demonstrating necessity of activity in targeted cell population for behavioral property a. Halobacterial HR i. Not ideal as optogenetic tool 1. Channel desensitizes (becomes unresponsive) b. Natranomonas pharonis (NPHR) gene i. Chloride channel pump activated by continuous yellow (589nm) light 1. Found to be more suitable in inducing inhibition in vivo ii. NpHR has undergone number of enhancements to improve membrane expression 1. eNpHR3.0 proven to be most effective in freely moving animal Optogenetic tool for modulating biochemical signaling 1. OPtoXR a. Type II fusion proteins b. A chimera of vertebrate rhodopsin and ligand gated G protein coupled receptors (GPCRs) c. Green light illumination  Gs / Gq / Gi signaling pathways i. ref slide 9 d. Slower optogenetic control (suffice to know) Using opsins as optogenetic tools 1. Optogenetic tools continue to be optimized for efficient transcription, expression, and safety 2. As the conductance of individual opsins is relatively low, it is critical to maximize the number of molecules that are properly integrated into the cytoplasmic membrane Effective delivery of optogenetic tools to target population of cells Delivery of light activated channels in specific populations of cells 1. Transgenic animals a. Breed animals to express optogenetic channels 2. Viral vector method a. Viral delivery 3. CRE-dependent expression a. Enzyme that manipulates genetic codes b. Mixture of transgenic and viral methods c. Only doable in mice Transgenic lines 1. Transgenic mouse / rat lines expressing ChR2 and NpHR are generated by microinjecting short transgene cassettes containing cell type specific or ubiquitous promotor and opsin gene 2. Strain is bred until a stable line is produced Transgenic approach 1. Good a. Uniformity of level of expression b. Distribution of genes 2. Bad a. Generating transgenic line is labor intensive i. Up to 1 year for a stable line b. Levels of transgene expression may be low i. Inadequate optical control Virus mediated approach 1. Uses ability of viruses to deliver genetic materials (opsins) into infected cells 2. Opsin gene is fused with a cell-type specific promoter, creating a recombinant virus 3. To minimize pathogenicity of the virus a. Viral DNA sequences for replication and production of the virus are separated from the viral DNA sequences for pathogenicity of the virus Viral vectors 1. Adeno-associated virus (AAV) and lentivirus are most widely used vectors due to a. Lack of pathogenicity b. Quick transgene expression c
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