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Original Paper: Adamantidis, A. R., F. Zhang, A. M. Aravanis, K. Deisseroth and L. de Lecea. 2007. Neural substrates of awakening probed with optogenetic control of hypocretin neurons. Nature 450: 420−424.

A common question in neurobiology is whether or not neurons in a particular brain region control a specific physiological or behavioral response in the animal. Recording neural activity with electrodes may show a correlation between activity of the neurons and the response, but that does not prove that those neurons control the response. The best evidence for whether specific neurons control a specific response is obtained by stimulating the activity of the neurons and observing—and thus proving—that the stimulation results in the response.

Dr. Karl Deisseroth’s lab at Stanford University developed a means of activating specific neurons with light. They called their method “optogenetics.” Algae have light-sensitive ion channels in their membranes. The scientists isolated the gene for one of these ion channels, rhodopsin (ChR2), and developed methods to modify the gene so that when it was introduced by viral infection into a specific brain area, it would be expressed only in the neurons of interest. Using this technique, the researchers introduced ChR2 into a specific group of cells in mouse brains that produce the neurotransmitter hypocretin. They also implanted optical fibers into the brains of these mice so they could shine light on these neurons and activate them. They hypothesized that these hypocretin-releasing neurons coordinated wakefulness.

Using electrodes, the scientists recorded the action potentials of the hypocretin neurons when they were stimulated with light. Figure A shows the response of a neuron to 1 second of light, and Figure B shows the response to 15-millisecond pulses of light. Each figure superimposes two trials.

The researchers recorded sleep and wakefulness in the mice, and they stimulated the hypocretin neurons when the mice were asleep. The control mice received the same viral infection treatment, but the virus did not include the ChR2 gene. The time between the stimulation and the animal awakening (sleep-to-wake latency) was determined for different stimulation frequencies. They repeated the same experiment in mice that were expressing the ChR2 gene but were injected with saline or high and low does of a drug that blocked the hypocretin receptor (HctR). Figure C shows the results of 15-ms light pulses over 10 seconds (1-30 Hz) and continuous light illumination for 10 seconds (ON). Asterisks indicate significant differences from controls.

Questions

Question 1

The 1-second light stimulation of the hypocretin neurons caused depolarization of their membrane potentials and a large increase in the rate at which they fired action potentials. These results support the concept that ChR2 is a Na⁺ channel and that light increases the Na⁺ permeability of that channel, depolarizing the membrane and bringing the membrane potential to above the threshold potential for firing of APs.

Question 2

The fact that the firing of the neurons followed precisely the trains of very short bursts of light (15 ms) indicates that the APs of the neurons were direct responses to the light.

Question 3

The light stimulation of hypocretinergic neurons expressing ChR2 in sleeping mice shortened the latency to awakening. The same light stimulation had no effect on the mice not expressing ChR2. Above a frequency of 1 flash per second, the awakening response was not altered by the frequency of stimulation.

Question 4

The fact that a blocker of the hypocretin receptor eliminated the awakening effect of the light stimulation supports the conclusion that the effect was mediated by the release of hypocretin by the stimulated neurons.