PLOS Student Bloggers Dan Albaugh and Jeremy Boringer discuss the ‘all optical’ symposium at the 2015 Society for Neuroscience (#SfN2015) conference.
Spatially restricted and temporally precise control of neural circuitry at the single cell level is considered something of a ‘Holy Grail’ in neuroscience research. In the past decade, huge advances have been made using light-sensitive proteins (called opsins) to allow for millisecond timescale control of individual neuron populations. Additionally, light-emitting (fluorescent) voltage or calcium sensors allow for direct visualization of neural activity, both spontaneous and in response to various experimental stimuli. At a recent SfN 2015 symposium, combining these two approaches in a so-called ‘all-optical’ experimental design was a topic of great interest and discussion.
The technical hurdles to such an approach are numerous, and involve genetically targeting the same populations of neurons with light-sensitive opsins as well as encoded activity sensors. Additionally, one must be able to record light signals from that population while simultaneously stimulating it with light. Consequently, many of the discussions surrounding such an approach were focused on recent advancements in microscope technology to allow such experiments to be conducted.
The symposium kicked off with Adam Cohen, professor of Chemistry and Chemical Biology at Harvard University. Dr. Cohen’s group has recently made great strides towards the fulfillment of an all-optical electrophysiology toolset with the development of Arch-3, a light-based indicator of membrane voltage. Now, his group has combined Arch-3 with ChannelRhodopsin-2 to allow for simultaneous optical stimulation and recording of voltage changes in neurons. This toolkit, cleverly termed the “Opto-Patch”, provides an optical near-equivalent to the patch clamp configuration in electrophysiology. Using ultra-fast recording methods (100,000 frames/second!), Dr. Cohen treated the symposium audience to a movie of opto-patch in action- visualization of an optically-driven action potential as it propagated out from the axon hillock. Critically, the optically-recorded membrane voltage signals acquired with opto-patch match those obtained with traditional electrophysiology, validating this promising toolkit for future neuroscientific studies, basic and translational alike.
In many cases, it may be advantageous to restrict optical stimulation to a subset of neurons, or even a single neuron within the imaging field. What are the optimal illumination methods to be combined with optogenetic tools? In the next talk, Valentina Emiliani of Paris Descartes University described two emerging approaches for optical cell targeting. The first approach, laser scanning, delivers a focal laser beam around a target cell (for example, in a spiral pattern) to optically-drive action potentials. This technique has been implemented with great success, although it unfortunately does not allow for multiple target neurons to be stimulated at the same time. Using an alternative method, termed parallel illumination, Dr. Emiliani described how multiple neurons can now be optically stimulated, selectively and in unison. The goal of parallel imaging in this context is to shine light over all neurons in a sample, but only allow for a subset of these neurons to be optically stimulated. This is achieved through the use of liquid crystal matrices, which can focus light on targeted cells while blocking off-target illumination of the sample. In sum, the field has now reached the point where a scientist can pick over a sample of neurons and selectively optically stimulate those interesting three in the middle and left corner.
If we can implement optical recording and optical excitement of the same individual cells, that would be the “dream experiment,” said Michael Hausser, professor of neuroscience at University College London. Dr. Hausser and his team have made significant progress towards this dream using a 2-photon microscopy approach. In an awake mouse, he was able to activate single cells in the barrel cortex (a brain region responsible for receiving sensory input from whisker stimulation). Furthermore, he could reliably stimulate up to 50 cells at once and record ‘ensemble’ activity (that is, groups of neurons that fired directly in response to whisker stimulation). He was able to identify ‘follower neurons,’ cells that didn’t respond to the stimulus directly but were activated by cells that did. Using this approach, he is able to tag cells based on their functional identity. For example, in response to the directionality of whisker deflection, he can find the ensemble (primary responders and follower neurons) of cells that respond to these specific changes in the barrel cortex. Importantly, this approach allows for identification of cell populations based on their functional properties, not just their genetic or anatomical properties.
Karl Deisseroth, an eminent figure in the optogenetics field, spoke next about applying these methods to study the neural circuit underpinnings of fear learning. This work began with the discovery by his team of an axonal projection from the anterior cingulate to the dorsal hippocampus (identified using a fluorescently tagged rabies virus that travels along axons). Deisseroth was quick to note the potential importance of a cortical input to dorsal hippocampus as a potential top-down regulator of hippocampal functions, such as fear learning. In line with this hypothesis, when the team used an optogenetic approach to cause the anterior cingulate to drive action potentials to the hippocampus after fear conditioning, they could cause the mouse to remember the fearful experience and exhibit a fear response. They also had a fluorescent calcium reporter in the hippocampus that emitted light when those neurons were activated. Turning on neurons with light in the anterior cingulate caused neurons in the hippocampus to light up in response, showing a functional connection. They further provided evidence that the anterior-cingulate connection to the hippocampus was the primary pathway important for this fear response because non-specific stimulation of the hippocampus did not cause the animal to experience fear.
In sum, the SfN symposium on all-optical interrogation of neural circuits provided a satisfying survey of the rapid progression in the burgeoning field of all-optical approaches for studying neural circuits. Improved opsins, fluorescent neural activity reporters, and novel light targeting methods were all discussed, as well as how these approaches can complement electrophysiological approaches to better understand neural circuit function in health and disease. The large and crowded conference hall was undoubtedly filled by scientists daydreaming about how all-optical toolsets can serve to the aid of their unique scientific questions. Indeed, it is difficult to imagine an area of neuroscience that could not benefit from such an experimental approach: from studies of neurodevelopment, psychiatric diseases, brain-machine interfaces and beyond.
Authors’ Note: A complementary minireview by the symposium speakers was recently published in the Journal of Neuroscience. This article expands upon many of the topics discussed in the symposium, and is a great resource for those interested in further exploring all-optical experimental designs for neural circuit studies.