BI2432 Fundamentals in Neurosciences - Neurotechniques: Optogenetics and Chemogenetics (Part 1)

BI2432 Fundamentals in Neurosciences: Neurotechniques

Why a Neurotechniques Module?

  • Neuroscience is a rapidly evolving field.
  • Progress often requires innovation in related disciplines like genetics, molecular biology, optics and imaging, and electronics.
  • This module covers diverse methods used in neuroscience labs and their contributions to important discoveries.

Lecture Schedule

  • March 7th, 13:10-14:00: Dr. Tim Wells - Transgenic animals
  • March 7th, 14:10-15:00: Dr. Maxime Assous - Optogenetics/Chemogenetics Part 1
  • March 14th, 9:00-9:50: Dr. Maxime Assous - Optogenetics/Chemogenetics Part 2
  • March 14th, 10:00-10:50: Dr. Jack Reddaway - Neuronal and glial cells morphology
  • March 21st, 13:10-14:00: Dr. Anurag Pandey - Anatomical Tracings
  • March 21st, 14:10-15:00: Dr. Frank Sengpiel - 1P/2P imaging of neuronal activity
  • March 28th, 13:10-14:00: Dr. Amanda Hornsby - Hippocampal neurogenesis and behaviors
  • March 28th, 14:10-15:00: Dr. Sean Wyatt - Neurotrophic Factors
  • April 4th, 13:10-15:00: Dr. Maxime Assous - Journal Club Seminar

Lecture 2: Optogenetics and Chemogenetics (Part 1)

  • Lecturer: Maxime Assous

Learning Outcomes for Optogenetics

  • Understand Opsins and their mechanisms.
  • Discuss the Cre-LoxP system and its use for delivering Opsins in Optogenetics.
  • Discuss the main advantages of optogenetics to manipulate neuronal subtypes and investigate the role of brain circuits.
  • Be able to design experiments using optogenetics.

Optogenetics Overview

  • Involves using light-sensitive proteins (Opsins).
  • Targets specific neuronal populations.
  • Allows turning on or off specific neurons in the brain.
  • Used to investigate the function of these specific neurons in circuits, behaviors, and diseases.
  • Combines genetic and optical methods to control neuronal activity.

Opsins

  • Major optogenetic tool.
  • Present in the retina.
  • Light sensitive.
  • Function in phototransduction (transform light into electrical signal).
  • Microbial Opsins are mostly used in Optogenetics.

Channelrhodopsin-2 (ChR2)

  • Derived from Chlamydomonas reinhardtii.

Optogenetics Modalities

  • Comparison with other methods:
    • Electrical
    • Physical
    • Pharmacological
    • Genetic
    • Optogenetic

Microbial Opsins

  • Different types respond to different wavelengths of light, allowing for activation or inhibition of neurons.
  • Examples include:
    • ChR (Channelrhodopsin): Allows influx of cations like Na^+ and Ca^{2+}.
    • BR/PR (Bacteriorhodopsin/Proteorhodopsin): Proton pumps.
    • HR (Halorhodopsin): Chloride pumps that hyperpolarize neurons.
    • OptoXR: Biochemical modulation.

Channelrhodopsin-2 Detailed

  • Light-gated cation channel (non-specific).
  • Sensitive to blue light (approximately 470nm).
  • Blue light induces a conformational change (all-trans to 13-cis).
  • This opens the pore of the ChR2 channel.
  • Cations flow into the cell, causing depolarization and action potential firing.
  • The channel returns to its original state within milliseconds, closing the pore.
  • Point mutations (e.g., H134R, E123T) affect the efficacy and kinetics of ChR2.

Channelrhodopsin-2 Stimulation

  • Can induce action potential firing at various frequencies (e.g., 5 Hz, 10 Hz, 20 Hz, 30 Hz) with blue light pulses (450-490 nm).

Channelrhodopsin-2 Variants

  • ChR2/H134R: A common and effective variant.
  • CHIEF: A chimera with an I170V mutation.

Halorhodopsin

  • 7-transmembrane protein (from halobacteria).
  • Light-activated chloride pump.
  • Sensitive to yellow/orange light (approximately 570-590nm).
  • Uses light energy to move chloride into the cell, causing hyperpolarization and silencing of neurons.
  • Multiple variants exist to increase efficiency and cellular localization.

Halorhodopsin and Channelrhodopsin Co-expression

  • Allows for both activation (using ChR2) and inhibition (using Halorhodopsin) of neurons.

Microbial Opsins – Peak Activation Wavelengths

  • A variety of opsins are available, each with different properties:
    • Fast Excitation:
      • ChR2 (Blue depolarizing)
      • ChETA variants
      • VChR1 (Red-shifted depolarizing)
    • Fast Inhibition:
      • NpHR
      • Arch
    • Step Function Opsins:
      • Bistable depolarizing

Cre-LoxP System

  • Used to get opsins specifically into a neuronal subtype.
  • Allows control over the location (and timing) of gene expression.

LoxP Sites

  • Specific 34-base pair sequences with an 8-base pair core and two flanking 13-base pair inverted repeats.
  • Recombination occurs within the core sequence.

Cre Recombinase

  • Catalyzes recombination between LoxP sites.
  • When LoxP sites are in the same orientation, Cre excises the DNA between them.
  • When LoxP sites are in opposite orientations, Cre flips the flanked DNA.
  • Floxing a gene allows it to be deleted, translocated, or inverted.
  • Discovered in bacteriophages (P1 bacteriophage) and not native to the mouse genome.
  • The orientation and location of LoxP sites determine the outcome of recombination.

Cre and LoxP Strains

  1. Cre-expressing strains:
    • Express Cre under a tissue-specific promoter.
  2. Inducible Cre strains:
    • Express a modified Cre that is non-functional until an inducing agent is administered (e.g., doxycycline, tetracycline, tamoxifen).
  3. LoxP-flanked (floxed) strains:
    • Contain LoxP sites flanking a critical portion of a target gene.
  4. Cre reporter strains:
    • Contain LoxP sites with visible marker proteins to trace Cre recombination success.

Examples of Specific Promoters

VGAT-Cre, VGluT2-Cre, PV-Cre, ChAT-Cre etc.

Adeno-Associated Viruses (AAVs)

  • Non-enveloped viruses with single-stranded DNA.
  • Members of the parvovirus family.
  • Genome is approximately 4.8 kilobases (kb).
  • Used to transduce cell lines/tissues with a gene of interest.

AAV Components

  • EF1a promoter
  • YFP (Yellow Fluorescent Protein)
  • ChR2 (Channelrhodopsin-2)
  • WPRE (Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element)

AAV Selectivity

  • Following injection, AAVs transfect neurons near the Cre sequence.
  • Phenotype-specific neurons express the gene of interest.
  • DIO (Double-floxed Inverted Orientation) and FLEX (Flip-Excision) switches are used for Cre-dependent transgene inversion.
  • Promoters need to be strong and ubiquitous (e.g., EF1a, Syn, Thy1, CMV, CAG).

Using AAVs for Optogenetics

  • Addgene provides AAV preparations of optogenetics plasmids.
  • Options include:
    • Opsins: Wild-type ChR2, ChR2/H134R, Arch and variants, etc.
    • Promoters: CAG, CaMKII, EF1a/nEF, Synapsin, etc.
    • Fluorophores: GFP, red/yellow/blue fluorescent proteins.
    • Activity: Cre-dependent, Flp-dependent, Constitutive.
    • Serotypes: AAV1, AAV2, AAV5, AAV8, AAV9, AAVrg

Experimental Approaches

  • Virus delivery system (floxed):
    • Uses a Cre promoter and a virus delivery system.
    • Advantages: Spatial selectivity, larger number of copies, combination of strategies.
  • Tg opsin-YFP Promoter:
    • Causes whole brain expression.
    • Advantages: Ubiquitous expression, consistent across subjects, non-invasive, immediate use.
  • Virus delivery system (non-floxed):
    • Naïve animals
    • Advantages: can maintain and expand colonies easily.

Next Week's Topics

  • Discuss Chemogenetics and the differences with Optogenetics.
  • Applications of optogenetic ex vivo and in vivo.
  • Journal Club: Analysis of a research article using these approaches. to learn how to read through the literature and extract the key information.