Review of Nat Neurosci︱Two-photon holographic optogenetics to detect neural coding

Source︱Privilege Science and Technology ︱Privy Council Editor in Chief︱Sizhen Wang In neuroscience, specific interventions on neurons are essential to understanding how neural circuits encode information and drive behavior
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Optogenetics has revolutionized the research paradigm of neuroscience by neuroscientists through its precise intervention in specific cell types and its rapid and reversible neuromodulation
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Graphical reconstruction in the intact brain using optogenetics
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Graphical reconstruction using single-photon excitation has great advantages, such as: the required hardware is relatively simple and inexpensive to use, relatively easy to stimulate a large number of neurons simultaneously, and the heat generated by the laser is low
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However, the scattering effect of the brain parenchyma severely affects the resolution of the images
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The current inability of single-photon optogenetics to reconstruct precise, cell-specific spatiotemporal activity patterns in highly dispersed brain tissue makes it challenging to specifically decode neuron-specific dynamics, for example using single-photon optogenetics Science is currently unable to address the effects of neuronal spiking frequency, neural synchrony, and neural networks on perception, cognition, and action
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However, two-photon optogenetics has high spatial and temporal precision in highly scattered brain tissue, and the expression of optogenetic opsins is relatively concentrated
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Graphically reconstructed two-photon optogenetics enables control over neuronal ensembles, and through synchronized two-photon functional imaging, neuroscientists can "read" and "rewrite" neural activity patterns with great precision
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 In August 2021, Hillel Adesnik and Lamiae Abdeladim of the Department of Molecular and Cellular Biology at UC Berkeley and the Helen Wills Neuroscience Institute published a paper in Nature Neuroscience entitled "Probing neural codes" "with two-photon holographic optogenetics" review article summarizes recent advances in two-photon holographic optogenetics and related technical challenges, and outlines the scope of experiments applicable to two-photon holographic optogenetics
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Two-photon holographic optogenetics could accelerate the pace of neuroscience and provide new insights into the causes of nervous system dysfunction
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Opsins for Two-Photon Optogenetics The biophysical properties of opsins are critical to the success of two-photon optogenetic holographic imaging experiments
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Traditional opsins such as ChR2 may require large amounts of opsin expression to evoke action potentials under two-photon stimulation due to their relatively low conductivity
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Therefore, one of the key issues in the progress of two-photon optogenetic holography is to optimize the opsin protein to make it more sensitive to light and have greater electrical conductivity
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Figure 1 Two-photon holographic optogenetics (Source: Adesnik H, Abdeladim L, Nat Neurosci , 2021) Chronos opsin single point mutation designed an ultrafast response opsin Chrome, Chrome’s current in response to light is four times that of Chronos , and therefore more efficient in activating neurons
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On the basis of Chrome, through engineering improvements, more responsive opsins ChroME2f and ChroME2s have been developed.
On the basis of greater responsiveness, the characteristics of fast dynamic response have been optimized
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In addition, the natural structure opsins CoChR and ChRmine have also been used in the research progress of two-photon optogenetics, but the kinetic characteristics of CoChR and ChRmine are relatively slow compared to the engineered opsins
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 In two-photon optogenetics, opsins that induce neuronal hypertrophy can inhibit specific types of neuronal activity
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GtACRs are a newly discovered class of anion channels, which themselves have extremely high electrical conductivity and high superficial properties
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The power of traditional optogenetics (such as eNpHR) photoinhibition is much larger than that of photoexcitation, because it is not clear when the neurons appear action potentials, and continuous photoinhibition is required, which may cause local temperature in the brain region.
too high
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And GtACRs excited by two-photon can solve this problem very well
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 Challenges for Two-Photon Optogenetics While two-photon optogenetics offers great advantages in precisely modulating the way neural activity is performed, several key issues must be addressed before expanding its utility and improving its precision
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 1.
How to effectively achieve single-cell resolution Although multiphoton imaging can achieve high optical resolution in the brain, a large number of experiments have found that in some cases, the resolution of multiphoton optogenetic imaging is greater than that of neuronal single cells resolution
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"Single-cell resolution" means that optogenetics activates only the target neuron without significant effects on other nearby neurons
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One way to address this problem is to use the Cre-off viral vector system to drive opsin expression only in target neurons, but it is still difficult to specifically intervene at the single-cell resolution scale
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The influence of neighboring neurons on target neurons cannot be ruled out
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To ensure single-cell resolution, off-target effects of photoactivation are also factors to consider
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 Achieving absolute accuracy at the single-cell scale also has several issues that must be considered: expressing opsins on the target neuron soma (rather than synapses) to drive action potentials, neuronal density in the brain parenchyma, intercellular opsins Heterogeneity of expression, and excitability between neurons of similar subtypes
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Despite the aforementioned challenges, neuroscientists have proposed several approaches to improve the fidelity of holographic imaging
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First, increasing the single-channel conductance of the opsin protein so that lower powers can be used to induce neuronal action potentials, thereby reducing the target deviation effect
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Second, opsins are concentrated in the soma to reduce activation of other neurons
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Third, reducing the expression of opsins makes excitable neurons more spatially distant from each other
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Fourth, excitation light that delivers less energy to each neuron induces neuronal action potentials, which may help maximize spatial fidelity
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 Figure 2 Improving the spatial fidelity of two-photon holographic optogenetics (Source: Adesnik H, Abdeladim L, Nat Neurosci, 2021) Second, how to capture more neuronal ensembles Two-photon laser scanning microscopy can image thousands of neurons The activity of a large number of neurons may be required for co-activation of a large number of neurons in some behaviors of animals, so the use of two-photon holographic optogenetics for research has great advantages
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There are two limiting factors for simultaneously controlling the number of neurons in a holographic field: the efficiency of the opsin and the power of the laser
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Combining multiple high-energy laser devices into a single microscope increases laser power, but can lead to localized hyperthermia in brain regions
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Therefore, optimizing opsins is an effective way to increase control neuron ensembles
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3.
The behavior of mesoscale two-photon holographic optogenetics animals depends on the interaction between multiple brain regions, and to understand how the activity pattern of one brain region leads to the specific activity pattern of the downstream region, it is necessary to accurately regulate light at the same time.
Read the data in these areas
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Mesoscale two-photon microscopy, which can achieve simultaneous imaging in an observation field greater than 5 mm, provides the feasibility of simultaneously measuring neuronal activity in multiple related brain regions, but the current imaging pixels need to be further improved
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 4.
Two-photon optogenetics in deep brain regions In mammals, traditional two-photon imaging is difficult to image deep brain regions due to the scattering of brain parenchyma
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Research protocols used in previous studies have included removal of brain tissue from targeted brain regions, and implantation of GRIN lenses or other optical components
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These protocols are equally applicable to two-photon optogenetics
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The current best imaging solution is to use three-photon excitation.
The depth of three-photon excitation can reach 1.
5 mm, which is very helpful for current structural imaging of moderate depths, but requires higher pulse energy, which may lead to excessive local temperature in the brain region.
high or tissue damage
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 5.
Two-photon optogenetics in freely moving mice So far, almost all two-photon holographic optogenetics studies require the fixation of the mouse's head under a microscope
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Long-range single-cell resolution multiphoton imaging and optogenetic modulation in freely moving animals is now highly feasible by using flexible fiber-optic systems
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The microscope can be coupled to a fiber optic bundle, which is typically further connected from the output face to a micro-GRIN lens
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Fig.
3 Research scheme of two-photon holographic optogenetics (Image source: Adesnik H, Abdeladim L, Nat Neurosci, 2021) Optimization of holographic optogenetic imaging The biggest disadvantage of holographic optogenetic imaging is the interference between nerve collections
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The precision of two-photon holographic optogenetics provides the ability to activate intervening arbitrary neural ensembles within the visual field, however, due to the large amount of noise in the experimentally measured midbrain regions, studies at the same level of perturbation repetition are required
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The easiest way to do this is to stimulate only one neuron at a time and calculate the neuron's effect on the neural network, since the net effect of most individual neurons is fairly small, multiple experiments are required, so only a single neuron can be detected per experiment A fraction of the meta impact
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What's more, intervention on a single neuron may not elicit any behavioral effects
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 Most recent studies of holographic optogenetics have focused on ensembles of neurons, where researchers can holographically image ensembles of neurons with the same common feature by classifying them according to their common responses to sensory or cognitive features
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In addition, the researchers computationally analyzed the neuron's physiological data to classify the neurons
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Optogenetic intervention was performed on the smallest set of neurons of the same type to determine the smallest set of neurons responsible for behavioral effects
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Figure 4 Two-photon optogenetics reveals neural ensembles driving behavioral effects (Source: Adesnik H, Abdeladim L, Nat Neurosci, 2021) Optimization of holographic optogenetic imaging 1.
Interpretation of neuronal spike firing and synchronization in visual perception The effect of neural synchronization in the visual cortex on sensory perception is one of the most well-known debates in sensory neuroscience
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Two-photon holographic optogenetics provides a way to resolve this debate
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First, action potentials can be evoked in ensembles of relevant visual cortical neurons, and the strength of the evoked action potentials is required to drive visual discrimination tasks
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Second, the exact same number of spikes were counted during the behavioral response time to study how different neural ensembles exhibited different degrees of synchrony, and to isolate neuron ensembles with the same synchrony
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In this way, it is possible to determine whether the spike discharge and synchronization of neurons in a unit time have an impact on visual perception
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2.
Resolving the effect of noise on sensory perception in the brain area Neurons in the cerebral cortex show high fluctuation in their discharges under the same stimulation
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The electrical signals of different neurons become noise in the target neurons of the study
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Although noise has been widely used to study the overall structure in neural circuits, the relevance of these noises to function is currently unclear
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Holographic optogenetics can conduct exploratory experimental studies on this issue
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In perception studies using holographic optogenetics, by arbitrarily controlling the excitation frequency of combinations of light stimuli, noise correlations can be reduced and the effects on behavior explored
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 3.
Investigate the effect of neural activity patterns on function Synaptic connections focus on the synaptic part of neurons, which is the smallest functional module in the activity of neural clusters
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Two-photon holographic optogenetics can intervene in the smallest functional modules of neural networks while tracking downstream neuronal activity and behavioral effects
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However, the most critical limitation of current two-photon optogenetics is the field of view, because many of the same neural clusters outside the field of view are still part of the neural network and can also affect behavior
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 4.
Exploring the effect of synaptic plasticity on neural networks during learning Rapid and dynamic changes of synapses are crucial to the formation of learning and memory
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Experience-dependent changes in synaptic activity underlie an important basis for encoding memory-related neural circuits
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Holographic optogenetics combined with calcium imaging can study the response of hundreds of neurons to learning and memory on large time scales ranging from milliseconds to days
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Figure 5.
Example of using multiphoton holographic optogenetics to process neural codes and plasticity rules (Credit: Adesnik H, Abdeladim L, Nat Neurosci, 2021) Conclusions and Discussions, Inspirations and Prospects Optogenetics has revolutionized over the past 15 years Although experimental neuroscience has been developed, traditional single-photon optogenetics methods are still difficult to solve basic problems of brain function
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Compared with single-photon optogenetics, two-photon optogenetics can provide sharper spatial resolution, which is beneficial to reconstruct precise neural activity patterns in specific behavioral paradigms
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Furthermore, two-photon holographic optogenetics has the potential to become the basis for therapeutic light-brain interfaces in the future, and utilize the precise resolution of holographic optogenetics to specifically intervene in targeted neuronal populations, potentially for patients with visual or hearing impairments The development of more realistic artificial prostheses and the potential to treat cognitive and affective disorders through precise intervention in closed-loop spaces
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 Link to the original text: https://doi.
org/10.
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