Neuroscience Lecture #11

Green Fluorescent Protein (GFP): Discovered by Shimomura in the jellyfish Aequorea victoria (aka Pecoria Victoria), causing its glow. (GFP) fluoresces green when exposed to blue light from a Blue Fluorescent Protein. Martin Chalfie enabled (GFP) expression in other animals (e.g., mice). Roger Tsien expanded this to Yellow Fluorescent Protein ((YFP)) and mCherry, enabling diverse visualization.

Opsin Receptors: Light-activated receptors existed in the 1970s.
Channelrhodopsins: In (2002/2003), researchers discovered in algae that proteins (channelrhodopsins) activate receptors in response to specific light colors.
- Channelrhodopsin-2 (CHR2): Activated by blue light, it opens channels allowing positive cations ( $Na^{+}$ , $Ca^{2+}$ , $H^{+}$ ) to enter neurons, leading to depolarization and action potential (excitation). Used for turning neurons ON.
- Halorhodopsin: Activated by yellow light, it opens channels allowing negative anions ( $Cl^{-}$ ) to enter neurons, leading to hyperpolarization and inhibition of action potentials. Used for turning neurons OFF.
Molecular Mechanism: The retinal molecule within the opsin protein changes from its cis to trans form when exposed to a specific light wavelength, causing the protein to open its channel/pore.

Genetic Code: The (DNA) sequences for these opsins (e.g., (CHR2), halorhodopsin) are known.
Plasmids: (DNA) sequences are inserted into plasmids, which typically include:
- A promoter: Determines the specific cell type or neuron where the opsin will be expressed (e.g., (CaMKII) promoter for excitatory neurons).
- The opsin gene sequence (e.g., (CHR2)).
- Often a fluorescent protein gene (e.g., (GFP) or (YFP)) for visualization to confirm successful transfection.
Viral Vectors: Modified viruses (e.g., AAV - adeno-associated virus, lentivirus, retrovirus) are used to deliver the plasmid into target cells, utilizing the virus's natural ability to infect cells. The viral capsid is retained, but harmful viral contents are removed.

Injection: A viral vector containing the opsin plasmid is injected into a specific brain region (or other tissue) using stereotactic surgery.
Fiber Optic Implantation: A fiber optic cable is implanted into the same area and secured to the skull (e.g., with dental cement).
Light Stimulation: A specific wavelength of light is shone through the fiber optic cable to activate (blue light for (CHR2)) or inhibit (yellow light for halorhodopsin) the genetically modified neurons.
Karl Deisseroth & Ed Boyden: Key figures in developing this technique by combining these discoveries.

Research: Precisely controlling specific neuronal circuits to study behavior, cognition (e.g., aggression, escape response in flies, Parkinsonian movement in rats, memory engrams in hippocampus).
Medical: Potential for treating diseases like Parkinson's (stimulating dopaminergic neurons), heart conditions (light-controlled pacemaker), and neurological disorders.
Addgene: A non-profit repository providing researchers access to various plasmids and viral services.
Ethical Implications: Concerns exist regarding the potential for manipulating personality, behavior, and memory, and the rapid pace of technological development outpacing public understanding and ethical regulation.