A rat explores a square, black box, where exposure to a new environment activates a specific collection of neuronal cells in the hippocampus, the brain’s memory center. As they are activated, these cells get “tagged” with channelrhodopsin, an ion channel that will render the circuit sensitive to activation by light. In a new, round, blue box, the rat is given a slight shock to the foot while light is delivered to the previously identified cells, re-activating the memory circuit from the black box. The rat freezes, learning to associate a fear memory with this stimulus. Later, the rat is returned to the square black box and freezes again, expecting a shock. Through a technique called optogenetics, Susumu Tonegawa’s lab has implanted a false memory in the rat.
Scientists have always utilized creative approaches to study the brain. Live animal, or in-vivo, studies look at the real time behavioral consequences of neural manipulation. For years, to study specific brain regions, researchers would damage the specific area and study the animal’s consequent behavior to understand the importance of the region. As technology improved, scientists became able to genetically alter neural circuits; a more precise and less risky approach in comparison to removing functional brain regions. These techniques allow scientists to target specific cell types, circuits, or whole areas without risking damage to other areas of the brain. Genetic manipulation, knockouts, and lesion studies prove to be compelling in neuroscience, however, most of these experiments are slow, invasive, and typically permanent. No simple way existed to get fast, precise control of neural activity that could be turned on and off, until Karl Diesseroth’s lab at Stanford University developed optogenetics.
What is Optogenetics?
Optogenetics combines light (‘opto’) with genetic modifications (‘genetics’) in order to create an experimental setup in live animals where modifications can be turned on and off as needed. The idea behind optogenetics is simple: make your subject (usually a rat or mouse) express an ion channel in the brain that is sensitive to light. Neurons transmit signals via the opening and closing of ion channels, thereby influencing their electrical activity. If light is delivered to one of these special ion channels, called an opsin, the cell can be activated or inhibited, depending on the needs of the experiment.
The first step in any optogenetic experiment is to induce expression of opsin. Light-sensitive channels were initially discovered in bacteria, but thanks to genetic engineering, it is possible to make almost any cell express these channels. There are several ways to achieve this; such as using genetically modified rodents, or by delivering a virus that contains DNA for the ion channel to the subject. Today, many types of opsins are available to suit the needs of any experiment. Two of the most common types are channelrhodopsin, a blue-light sensitive excitatory ion channel, and halorhodopsin, a green-light sensitive inhibitory channel. For example, if there was an animal expressing both Channelrhodopsin and Halorhodopsin, delivering blue light would cause its neurons to fire while delivering green light would inactivate them. By utilizing different genetic characteristics, opsins can be optimized for specific cell types, brain regions, or activation/inactivation speeds.
After an animal expresses the opsin, researchers implant a fiber optic cable in the brain. This cable attaches to a light source that delivers light at the wavelength responded to by the channel, usually blue, green, or red, depending on the opsin. The light source, usually a laser or an LED, switches on and off and ion channels operate within milliseconds. This means that these circuits can be turned on and off completely, literally with a flick of a switch.
Memory Studies
One of the most innovative uses of optogenetics is in memory studies. A common behavioral test known as conditioning reveals the mechanisms behind memory systems, and relies on the hippocampus. Conditioning results in associative learning, where an animal earns to associate two independent stimuli. A subject’s ability to predict a second stimulus after exposure to the first indicates if associative memory was affected by an experiment. A scientist will train the subject to two stimuli, such as a high-pitched tone (the “conditioned” stimulus which normally would not evoke a behavioral response) and a puff of air to the eyes (a response-evoking “unconditioned” stimulus). If learning occurs, after training, the subject should automatically react to the unconditioned stimulus, in this case by closing the eyes upon hearing the high-pitched tone. This is called eyeblink conditioning.
The process by which the two stimuli are associated is called long term potentiation (LTP). When learning occurs, synapses at neurons within a memory circuit strengthen, resulting in transmission of more neurotransmitters and increased sensitivity of the postsynaptic neuron. LTP explains how associations are made during conditioning experiments. After a period of time, depending on the strength of LTP and the degree to which a memory has been reinforced, synapses may be desensitized and weakened through a process called long term depression (LTD). This allows unnecessary connections to be lost so that new memories can be made and stored, essentially how forgetting works.
But what does this have to do with optogenetics? If a scientist wants to study the importance of a certain brain region or synaptic connection in learning, they can perform an experiment in which they inhibit or activate the area of interest only during conditioning. If an area gets optogenetically modified during a behavioral experiment and learning outcomes are affected, this demonstrates precisely an important link between the area and learning. Experiments like these enable the animal to have a normal life outside of the experiment, and help to establish causal relationships between synaptic processes and memory.
How UC San Diego Scientists Engineer Memories
One of the most famous optogenetic memory experiments was conducted by Sadegh Nabavi in Roberto Malinow’s lab here at UC San Diego. Animals were conditioned using a classical conditioning method: a tone paired with foot shocks, eliciting an easily measurable fear response. Upon hearing the tone, subjects produce a fear response, indicating learning occurred. This is a common and simple technique, but the Malinow lab did something new: they replaced the tone with excitatory light delivery to the auditory cortex, activating the auditory stimulus response without the actual stimulus being present. Pairing optogenetic stimuli with a fearful stimulus induced LTP in the animal in the same way that a tone typically would. Furthermore, inducing LTD by optogenetic stimulation resulted in a loss of the fear response. Synapses can be desensitized by weak, prolonged exposure to the conditioning stimulus, resulting in synaptic depression and loss of the memory association. By precisely activating and inactivating specific neuronal assemblies, the inner workings of memory circuits are revealed.
How are Optogenetics Being Used Today?
As optogenetics becomes easier, more affordable, and more widely used in labs, our understanding of brain circuitry continues to rapidly develop under more precise and innovative techniques. One of the most straightforward ways to study whether a certain synapse, cell, circuit, or region within the brain is needed for learning is to inactivate it and identify whether memories are still formed. The precise temporal control offered by optogenetics allows experimenters to use this general idea while opening up the field to even more questions to be uncovered. For example, perhaps a scientist wants to know exactly what time window is important for memory formation. The experimental procedure would be as follows: inhibiting the hippocampus (or any brain region involved in learning) during the training period for the conditioned stimulus, unconditioned stimulus, or the period in between. This can be easily done with an inhibitory opsin, like halorhodopsin, and brief light delivery at a chosen time point. The lack of a conditioned response after light delivery at one of these experimental timepoints would indicate that a memory has not been formed, and that LTP is dependent on that timepoint.
New applications for optogenetics are being developed every day by labs across the country. Some labs are enhancing memories to treat disorders like Alzheimer’s disease, while others have successfully implanted false memories and used induced LTP to recover “lost” memories. Though some time remains before these technologies are ready for human applications, the budding field of optogenetics offers innovative research leading to promising therapeutics for memory disorders and beyond.
Sources:
https://unsplash.com/photos/human-brain-toy-IHfOpAzzjHM
https://www.nature.com/articles/nature11028
https://www.nature.com/articles/nprot.2009.226
https://www.nature.com/articles/nature13294
https://www.jove.com/t/51483/in-vivo-optogenetic-stimulation-of-the-rodent-central-nervous-system