Optogenetic control of cells and circuits

Abstract

The absorption of light by bound or diffusible chromophores causes conformational rearrangements in natural and artificial photoreceptor proteins. These rearrangements are coupled to the opening or closing of ion transport pathways, the association or dissociation of binding partners, the enhancement or suppression of catalytic activity, or the transcription or repression of genetic information. Illumination of cells, tissues, or organisms engineered genetically to express photoreceptor proteins can thus be used to perturb biochemical and electrical signaling with exquisite cellular and molecular specificity. First demonstrated in 2002, this principle of optogenetic control has had a profound impact on neuroscience, where it provides a direct and stringent means of probing the organization of neural circuits and of identifying the neural substrates of behavior. The impact of optogenetic control is also beginning to be felt in other areas of cell and organismal biology.

Figure 1

Cell biological processes that have been controlled with the help of optogenetic actuators.

Figure 2

Optical control of three types of photoreceptor proteins. (a) Natural photoreceptor protein with bound chromophore. In the example depicted, absorption of a photon of 480-nm light causes isomerization of a retinal chromophore, which triggers a conformational change in a retinylidene photoreceptor. The lit state reverts to the ground state via absorption of a photon of a different color (580 nm), thermal relaxation (Δ), or enzymatic back conversion that requires ATP hydrolysis. (b) Artificial photoreceptor protein with a photoisomerizable tethered ligand (PTL). Absorption of a photon of 380-nm light causes trans-to-cis isomerization of an azobenzene tether bearing an allosteric effector. Isomerization brings the effector in contact with its binding site on the receptor, which changes conformation. The lit state reverts to the ground state via thermal relaxation (Δ) or absorption of a 500-nm photon. (c) Artificial photoreceptor protein and caged effector. In the example depicted, attachment of a dimethyl-nitrobenzyl group blocks the biological activity of an effector. Absorption of a 355-nm photon causes photolysis of the caging group and release of the biologically active effector. Binding of the effector to its cognate receptor induces a conformational change in the receptor protein. Photolysis is irreversible.

Figure 3

Control of electrical signals. (a–c) Pulse illumination of mammalian neurons expressing the optically gated channels LiGluR (a), ChR2 (b), and P2X₂ (c) elicits single action potentials. Membrane potential changes recorded during four repetitions of an optical stimulus at 1 Hz are overlaid; traces are aligned to the onset of illumination. Optical stimulation regimes are indicated by colored bars at the bottom of the traces. (a) Continuous green (500-nm) illumination is paused, and a 1-ms pulse of 380-nm light is applied to gate open LiGluR. (b) A 20-ms pulse of 473-nm light activates ChR2. (c) A 5-ms pulse of 355-nm light uncages ATP to activate P2X₂. Shaded circles in the background symbolize the single-channel conductances of the actuator molecules and the approximate openings of a population of channels in response to light. Recordings in panel a were obtained from transfected hippocampal neurons in dissociated culture (courtesy of S. Szobota and E. Isacoff). Recordings in panels b and c were obtained from inhibitory neurons in neocortical slices, which were harvested from knock-in mice carrying otherwise identical cassettes for expression of the respective actuators at their GT(ROSA)26Sor loci (Kätzel et al. 2011). (d,e) Pulse illumination of pyramidal neurons in neocortical slices obtained from knock-in mice carrying otherwise identical cassettes for expression of the respective actuators at their GT(ROSA)26Sor loci (D. Kätzel & G. Miesenböck, unpublished data). Both ChR2 and P2X₂ are effective in driving spiking in inhibitory neurons (b,c), but ChR2 causes only subthreshold depolarizations in pyramidal cells (d), presumably because of its small single-channel conductance (b). P2X₂, whose single-channel conductance is two to three orders of magnitude larger than that of ChR2 (c), also drives action potentials in cortical pyramidal neurons (e).

Figure 4

Control of behavior. The circuit diagrams on the right present an extremely simplified view of the brain, illustrating targets of optogenetic intervention (the circuit elements in yellow). Signals from external and internal sensors are used to construct internal representations of the animal’s state. Behavior is generated when states are mapped onto actions and the resulting action representations recruit motor systems. The probabilistic rules determining which states are mapped onto which actions are subject to short- and long-term modulation. (a) Flies sense ambient CO₂ levels as an index of stressful overcrowding. Optogenetic activation of CO₂-sensing neurons creates the illusion of crowded conditions and elicits avoidance behavior (Suh et al. 2007). (b) Pheromonal, gustatory, and visual signals indicate the presence of a potential mating partner. In the male brain, the state “presence of receptive female” is mapped onto the action “courtship,” which leads to the activation of a motor program involving a unilateral wing beat, the so-called courtship song. (c) In the female brain, the state “presence of receptive female” is not mapped onto the action “courtship.” However, the motor program for male-specific courtship is still present in the female, as it can be recruited optogenetically, bypassing sensory input altogether (Clyne & Miesenböck 2008).

Figure 5

Reprogramming of behavior. The circuit diagrams on the right present an extremely simplified view of the brain, illustrating targets of optogenetic intervention (the circuit elements in yellow). Signals from external and internal sensors are used to construct internal representations of the animal’s state. Behavior is generated when states are mapped onto actions and the resulting action representations recruit motor systems. The probabilistic rules determining which states are mapped onto which actions are subject to short- and long-term modulation. (a) When a fly approaches odor No. 5, an attractant, dopaminergic modulatory neurons carrying aversive reinforcement signals are optogenetically activated. (b) This intervention results in a lasting remapping of the state “presence of odor No. 5” onto the action “avoidance”: An aversive olfactory memory has been programmed artificially (Claridge-Chang et al. 2009).

Figure 6

Analysis of synaptic connectivity. (Left panel) One particular class of neuron (red) in the schematic tissue slice on the left is engineered to express an optogenetic actuator. Transmembrane currents in a potential target cell (green) are recorded while a laser beam scans the tissue slice. If an illuminated spot in the slice harbors an optogenetically activated cell that is connected to the recorded neuron, a synaptic current appears in the recording (top right). The magnitudes of the currents evoked at different locations are color coded to produce the input map (right panel).