Scared by the light

WHO could have guessed that a protein isolated from pond scum would transform the way researchers investigate the brain? The protein, called channelrhodopsin (ChR), is found in algae and other microbes, and is related to the molecule in human photoreceptors that captures light particles. Both versions control the electrical currents that constantly flow in and out of cells; one regulates the algae's movements in response to light, the other generates the nervous impulses sent along the optic nerve to the brain. Unlike its human equivalent, the algal ChR controls the currents directly because it forms a pore that spans the cell membrane. When expressed in neurons, it renders the cells sensitive to light, and they can be switched on or off very precisely using lasers.

This discovery led to the emergence of a new field called optogenetics. Early studies showed that the technique can be used to control the behaviour of small organisms such as nematode worms and fruit flies. Last year, Karl Deisseroth's group at Stanford University demonstrated, for the first time, that it can also be used to control reward and motivation behaviours in mice. Josh Johansen of the Center for Neural Science at New York University and his colleagues have now taken this one step further. Working in collaboration with Deisseroth, they show that optogenetics can also be used to induce a simple form of associative learning called fear conditioning.

Fear conditioning typically involves associating an innocuous stimulus such as an audible tone with an aversive stimulus such as electric shock. With repeated pairings, the animal quickly learns an association between the two, and subsequently expects to receive a shock whenever it hears the tone. As a result, it will exhibit a fear response (freezing) when the innocuous stimulus is presented alone.  The initial association between the two stimuli is thought to be due to synaptic plasticity in the amygdala - the inputs related to each stimulus converge on the same group cells, causing the connections between them to be strenthened. As a result, a memory associating the innocuous stimulus with the aversive one is encoded, but this quickly fades, or becomes extinct, if the innocuous stimulus is then repeatedly presented alone.

Johansen and his colleagues substituted the electric shock with pulses of laser light, delivered to the lateral nucleus of the amygdala through a fibre optic cable. This was done in three groups of rats - in one, the light pulses were delivered at the same time as a tone; in another, just before; and in the third, just after. 24 hours later, they played the tone back, and tested the animals' responses to it. The first group of rats froze when they heard it,  biut the other two did not, showing that the light pulses were sufficient to induce fear conditioning, but only when delivered at the same time as the tone. This supports the view that associative learning occurs when inputs representing the two stimuli coincide on the same neurons.

The observed fear responses were, however, significantly smaller than those seen when electric shocks are used, or when the central nucleus of the amygdala is electrically stimulated. This might be because the optical stimulation activated cells in the lateral nucleus indiscriminately, interfering with proper encoding of the memory. A more likely explanation is that the lateral amygdala alone is insufficient to generate a full fear response, and that other components of the brain's fear circuitry are also required. One of these other components is the central nucleus of the amygdala, whose role in fear conditioning overlaps with, but is distinct from, that of the lateral mucleus. 

"The central nucleus is especially involved in controlling responses," says senior author Joseph LeDoux, "while the lateral nucleus is especially involved in sensory processing and  association [between the two stimuli], and is the initial site of plasticity. The central nucleus is also a site of plasticity, but seems to be downstream, which is why we focused on the lateral nucleus." LeDoux's group is now introducing ChRs into different cell types in the amygdala, in order to examine the contribution of each to fear conditioning. They are also investigating the effects of various neuromodulators on synaptic plasticity in the amygdala.   

Optogenetics is still in its infancy, and will undoubtedly become more advanced with time, enabling researchers to probe the cellular mechanisms of fear conditioning in greater detail. Indeed, a group from the Howard Hughes Medical Institute has just reported using the technique in combination with two-photon microscopy, not only to target single cells in the hippocampus, but also to selectively activate distinct subcellular compartments such as the axon and dendrites. Such advances will eventually provide a more detailed understanding of how the amygdaloid nuclei are connected to one another and to other parts of the fear circuit, as well as how information related to fear conditioning is processed by individual cells and neural networks. 

Related:

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Johansen, J., et al. (2010). Optical activation of lateral amygdala pyramidal cells instructs associative fear learning. Proc. Nat. Acad. Sci. DOI: 10.1073/pnas.1002418107.