What's the science?
Different information in the brain is encoded by the firing of specific neurons, but also by the timing, rate of that firing, and synchrony of firing with other neurons. Therefore, in order to understand and control neural activity, we must be able to control each of these parameters. The ideal experimental technique would have both high spatial and high temporal resolution over a large volume of brain tissue, and be able to create as well as remove existing activity. Two-photon optogenetics is a technique used to stimulate specific neurons: genes are inserted into neurons, which allows these neurons to produce light-sensitive proteins (opsins), and then light is shone on these neurons, generating action potentials. However, there are limitations to how precise the timing of stimulation, number of neurons stimulated and location of stimulation can be with optogenetics. This week in Nature Neuroscience, Mardinly and colleagues engineered a new technique that allows for precise temporal and spatial control of neural activity using special opsins and holography (a technique that can illuminate the entire cell body/soma of a neuron) to ‘write’ neural activity, along with simultaneous volumetric calcium imaging to ‘read’ neural activity.
How did they do it?
They used an experimental setup that incorporated holography, which involves lasers and bending light to illuminate multiple specific neurons in 3D space, allowing for fast excitation of neurons at precise spatial locations.
Experiment Set 1 → First, they studied the characteristics of several opsins with the goal of developing two opsins: one that could generate large excitatory currents quickly to activate cells and one that generated large inhibitory currents to suppress cells. Using patch clamping (in anesthetized mice) and brain slice recording techniques in mice, they tested several opsins for the amplitude and kinetics of the photocurrent elicited. They mutated the chosen opsins to optimally generate or suppress activity. They then tested the precision and speed with which neurons with this opsin elicited action potentials.
Experiment Set 2 → Next, they used holography (to stimulate or suppress neurons) and calcium imaging (to ‘read’ (i.e. record) the resulting neural activity) at the same time. Mice who expressed neurons with both chosen opsins ran on a treadmill with their heads fixed in place, and neurons in the primary somatosensory cortex were activated or suppressed (depending on the opsin) using a holographic technique. Neurons were activated or suppressed one at a time, and later in random groupings, to test whether the activity of multiple different types of neurons could successfully be altered at the same time.
What did they find?
Experiment Set 1 → After testing several excitatory opsins, they mutated the fastest opsin tested to create a highly potent but still equally fast version, that they call ST-ChroME. After genetic mutation of this opsin they observed that it required low laser power (i.e. light shone) to produce action potentials with short latency and low temporal variability (which is desirable) during brain slice recordings. Using patch-clamp recordings, they found that neurons with ST-ChroME exhibited action potentials reliably (89% of the time). ST-eGtACR1 was the opsin found to be the most effective at suppressing neural activity. Using patch-clamp recording, the firing rate of neurons with ST-eGtACR1 was reduced to 8% of normal firing rate during holographic suppression.
Experiment Set 2 → Next, mice ran on a treadmill and neuronal activity was simultaneously stimulated and recorded. Neurons (with ST-ChroME) activated one at a time produced reliable calcium signals, indicating they could be reliably activated in vivo. In vivo optical suppression resulted in a reliably reduced calcium response. Finally, when entire groups of neurons were stimulated, calcium signalling was as expected, indicating neurons of different sizes or functions can be activated simultaneously. Up to 50 neurons could be activated at the same time, allowing for thousands of light-evoked action potentials per second.
What's the impact?
In this study, optogenetic techniques (newly engineered opsins—proteins activated by light) were combined with holography (bending of light to be distributed over a 3D volume) to stimulate neurons precisely. By combining holography with calcium imaging, the authors facilitated spatially and temporally precise simultaneous stimulation and recording (‘write’ and ‘read’) of neuronal activity respectively. Holography allowed for specific neurons at various different locations in a 3D volume of the brain to be activated at the same time. This study expands on previous optogenetic techniques which lacked a high level of spatial and temporal precision and will help us to perform a wide variety of experiments where the activity of multiple neurons can be altered with high spatial and temporal precision.
A. R. Mardinly et al., Precise multimodal optical control of neural ensemble activity. Nature Neuroscience (2018). Access the original scientific publication here.