{Optogenetics

1979

“The major challenge

facing neuroscience is

the need to control one type

of cell in the brain while

leaving others unaltered.”

1971

Bacteriorhodopsin

2002

Channelrhodopsin

2005“A lot of people

thought about it,

but nobody

was crazy enough to try it…we

were.”

A Brief Recap

• Optogenetics is a technique which allows researchers to turn certain regions of the brain on and off without damaging their function in the long term

• This has been a major breakthrough in the field of Neuroscience as it has advantages over previous techniques of studying brain function:

• Loss of function studies using lesions resulted in permanent damage to brain tissue and little re-usability of test animals

• Stimulation of brain areas with electrodes were limited by the inability to target single cells or specific cell type populations

How Does It Work?

• The genes for light-activated ion channels are introduced to a population of cells by a human engineered virus

• Which cells express these light-sensitive channels depends on the promoter region of the inserted DNA sequence

• Cells which contain a promoter that can recognize the promoter sequence will express these channels while cells that lack a promoter specific for the sequence will not

Beyeler et al. 2014

How Does It Work? Cont.

• Once the genes have been inserted, it can take 1-2 weeks for them to be fully expressed

• When studies are ready to be run, a fiber optic cable is surgically attached to the top of the skull or inserted near the brain area of interest depending on how close it is to the surface of the brain

• The channels, and in turn the neurons in which they are embedded, can now be controlled by light from the optic cable

Beyeler et al. 2014

The Channels

Depolarizing (Excitatory) Hyperpolarizing (Inhibitory)

Tracing Engrams with Optogenetics

Ramirez et al. 2014

Engram?

• The German zoologist Richard Semon coined the term in 1921

• According to Semon, an engram was the biological conceptualization of a memory

• He may have inspired the initial search for the basis of memory in the brain

Richard Wolfgang Semon

Ramirez et al. 2014

How Can We Trace Engrams? With the Help of IEG’s of Course!

• An IEG is an Immediate Early Gene

• It has been hypothesized that IEG’s are expressed in neurons involved in the formation of a memory

• Blocking the transcription of a specific IEG, c-fos, impairs memory in mice

c-fos (In Red)Ramirez et al. 2014

• With this information in mind the solution to tracing a memory seems simple at first: Attach a c-fos promoter to a Channelrhodopsin gene

• New memories are being made all the time though. How are researchers to distinguish the memory they want from other memories formed?

The Solution

• Researchers inserted two genes into the Dentate Gyrus of the Hippocampus, a region critical for memory formation.

• tTA with a c-fos promoter (transgenic)• ChR2-EYFP with a TRE promoter

(virus)

• TRE is activated by tTA but also blocked by the antibiotic doxycycline

• Therefore to prevent unwanted ChR2-EYFP expression until testing, researchers put test animals on a diet of doxycycline infused food

Ramirez et al. 2014

tTAC-fos

DOX

ChR2-EYFPTRE

Ramirez et al. 2014

The Experiment• Researchers wanted to know if they could

trace a memory engram with ChR2-EYFP in one context (in this case meaning environment) and then activate that memory with light in a completely unrelated context

• In this case the target memory would be that of the environment in which fear conditioning took place

• Fear conditioning is a classic method of studying behavior in which a stimulus (in this case the environment) is associated with pain, usually in the form of a quick electric shock

• When presented with that environment or stimulus in later tests, animals exhibit a behavior called “freezing” in which they pause in expectation of a shock

Ramirez et al. 2014

The Experiment Cont.

• Mice were taken off of a doxycycline diet and underwent fear conditioning in Context B

• After fear conditioning a doxycycline diet was immediately resumed

• Mice were then placed in Context A and blue light was delivered to the DG. This induced freezing despite the mice not being in a fear conditioning environment.

• Researchers had successfully traced an engram and induced recall out of context.

“This experiment directly

tests the hypothesis that the

c-fos- expressing cells in the

hippocampus activated

during training are sufficient

for memory recall”

– Ramirez et al. 2014

Ramirez et al. 2014

Do Engram Cell Populations Overlap?

• Mice were taken off of a doxycycline diet and placed in Context A (The memory of context A was traced)

• Afterwards a doxycycline diet was immediately resumed

• While on doxycycline, the mice underwent fear conditioning in Context B (Not traced)

• If the cells responsible for the memories of A and B overlap then it would be expected to see freezing when stimulated with blue light.

• No freezing occurred

“Accordingly, we propose

that these cells form a

cellular basis of a memory

engram, and that two

different contexts are

parsed out as independent

experiences represented by

independent neuronal

ensembles in DG.”

– Ramirez et al. 2014 Ramirez et al. 2014

Ramirez, Steve, Susumu Tonegawa, and Xu Liu. "Identification and Optogenetic Manipulation of Memory Engrams in the Hippocampus." Frontiers in Behavioral Neuroscience 7 (2014)

Beyeler, Anna, Christine A. Eckhardt, and Kay M. Tye. "Deciphering Memory Function with Optogenetics." Progress in Molecular Biology and Translational Science 22 (2014): 341-90. Web.