Double-μPeriscope, a tool for multilayer optical recordings, optogenetic stimulations or both
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Evaluation Summary:
This Tools and Resources article presents an innovative method for simultaneously stimulating and imaging two cortical layers in tandem while causing minimal damage to brain tissue. The method substantially builds on existing methods in several ways, while still pinpointing the limitations of existing methods that are overcome in this new approach. Three well-described sets of experiments demonstrate the method's reliability and versatility, and highlight its promise in tackling big questions about cortical microcircuit functions.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Intelligent behavior and cognitive functions in mammals depend on cortical microcircuits made up of a variety of excitatory and inhibitory cells that form a forest-like complex across six layers. Mechanistic understanding of cortical microcircuits requires both manipulation and monitoring of multiple layers and interactions between them. However, existing techniques are limited as to simultaneous monitoring and stimulation at different depths without damaging a large volume of cortical tissue. Here, we present a relatively simple and versatile method for delivering light to any two cortical layers simultaneously. The method uses a tiny optical probe consisting of two microprisms mounted on a single shaft. We demonstrate the versatility of the probe in three sets of experiments: first, two distinct cortical layers were optogenetically and independently manipulated; second, one layer was stimulated while the activity of another layer was monitored; third, the activity of thalamic axons distributed in two distinct cortical layers was simultaneously monitored in awake mice. Its simple-design, versatility, small-size, and low-cost allow the probe to be applied widely to address important biological questions.
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Author Response
Reviewer #1 (Public Review):
The goal of this Tools and Resources article was to present a new method for optogenetic stimulation and optical imaging at the same time in two different cortical layers in vivo, and through 3 sets of experiments, highlight the promise and wide applicability of this method.
The method itself presents an elegant solution to several outstanding drawbacks among the many recent innovations in these lines of methodology, including high expense, lack of specificity and excessive brain tissue damage. The paper provides what I believe to be a fair account of the capabilities and limitations of existing methods and a clear description of how the new method builds on and overcomes these.
The three sets of experiments work well because they demonstrate reliability and feasibility in replicating …
Author Response
Reviewer #1 (Public Review):
The goal of this Tools and Resources article was to present a new method for optogenetic stimulation and optical imaging at the same time in two different cortical layers in vivo, and through 3 sets of experiments, highlight the promise and wide applicability of this method.
The method itself presents an elegant solution to several outstanding drawbacks among the many recent innovations in these lines of methodology, including high expense, lack of specificity and excessive brain tissue damage. The paper provides what I believe to be a fair account of the capabilities and limitations of existing methods and a clear description of how the new method builds on and overcomes these.
The three sets of experiments work well because they demonstrate reliability and feasibility in replicating previous findings from older techniques such as the phenonmenon of 'backpropagation-activated calcium spike firing' and net inhibitory influence of layer 2/3 cells on layer 5 cells, while also extending beyond those findings by verifying that some effects generalise to other areas than previous observations - the layer 2/3-5 interaction previously seen in primary somatosensory is here extended to motor cortex - and uncovering interesting phenomena that are relatively unexplored to date - the great variability in the degree of mirroring of activity in two layers receiving axonal input from the same thalamic area.
The method presents exciting possibilities for the fine-grained study of cortical microcircuits and how they enable perception and cognition and relate to behaviour. The simplicity and low cost of the solution opens it up to a wider range of laboratories globally, and its low-profile imprint on the cortex ensures that it most likely reflects activity of normal, intact, rather than damaged, cortical tissue.
We thank Reviewer #1 for recognizing “exciting possibilities” and advantages of our method.
Reviewer #2 (Public Review):
This manuscript reported a new approach to conduct neural activity imaging and manipulation in two different cortical layers. Two periscopes, each constructed from a micro-prism, a GRIN lens and a multi-mode fiber, could be inserted to the brain at different depths, and each can either perform imaging or optogenetics. The authors demonstrated a few applications: stimulation of L5 soma and superficial layer dendrites to evoke backpropagating action potential; optogenetically stimulating cells in L2/3 and observing response in L5 to investigate interaction between cells in two different layers; and simultaneously recording axon terminals from posteromedial thalamic nucleus at two different depths in cortex. This works combines the ideas of fiber photometry to access deep layers and using microprism to turn the optical field of view by 90 deg.
Major strengths
• Using microprism to perform layer specific imaging or optogenetics.
• Low cost
• Demonstrations of a few applications that require layer specific imaging and optogenetics.
We thank Reviewer #2 for recognizing these strengths.
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Evaluation Summary:
This Tools and Resources article presents an innovative method for simultaneously stimulating and imaging two cortical layers in tandem while causing minimal damage to brain tissue. The method substantially builds on existing methods in several ways, while still pinpointing the limitations of existing methods that are overcome in this new approach. Three well-described sets of experiments demonstrate the method's reliability and versatility, and highlight its promise in tackling big questions about cortical microcircuit functions.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The goal of this Tools and Resources article was to present a new method for optogenetic stimulation and optical imaging at the same time in two different cortical layers in vivo, and through 3 sets of experiments, highlight the promise and wide applicability of this method.
The method itself presents an elegant solution to several outstanding drawbacks among the many recent innovations in these lines of methodology, including high expense, lack of specificity and excessive brain tissue damage. The paper provides what I believe to be a fair account of the capabilities and limitations of existing methods and a clear description of how the new method builds on and overcomes these.
The three sets of experiments work well because they demonstrate reliability and feasibility in replicating previous findings from …
Reviewer #1 (Public Review):
The goal of this Tools and Resources article was to present a new method for optogenetic stimulation and optical imaging at the same time in two different cortical layers in vivo, and through 3 sets of experiments, highlight the promise and wide applicability of this method.
The method itself presents an elegant solution to several outstanding drawbacks among the many recent innovations in these lines of methodology, including high expense, lack of specificity and excessive brain tissue damage. The paper provides what I believe to be a fair account of the capabilities and limitations of existing methods and a clear description of how the new method builds on and overcomes these.
The three sets of experiments work well because they demonstrate reliability and feasibility in replicating previous findings from older techniques such as the phenonmenon of 'backpropagation-activated calcium spike firing' and net inhibitory influence of layer 2/3 cells on layer 5 cells, while also extending beyond those findings by verifying that some effects generalise to other areas than previous observations - the layer 2/3-5 interaction previously seen in primary somatosensory is here extended to motor cortex - and uncovering interesting phenomena that are relatively unexplored to date - the great variability in the degree of mirroring of activity in two layers receiving axonal input from the same thalamic area.
The method presents exciting possibilities for the fine-grained study of cortical microcircuits and how they enable perception and cognition and relate to behaviour. The simplicity and low cost of the solution opens it up to a wider range of laboratories globally, and its low-profile imprint on the cortex ensures that it most likely reflects activity of normal, intact, rather than damaged, cortical tissue.
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Reviewer #2 (Public Review):
This manuscript reported a new approach to conduct neural activity imaging and manipulation in two different cortical layers. Two periscopes, each constructed from a micro-prism, a GRIN lens and a multi-mode fiber, could be inserted to the brain at different depths, and each can either perform imaging or optogenetics. The authors demonstrated a few applications: stimulation of L5 soma and superficial layer dendrites to evoke backpropagating action potential; optogenetically stimulating cells in L2/3 and observing response in L5 to investigate interaction between cells in two different layers; and simultaneously recording axon terminals from posteromedial thalamic nucleus at two different depths in cortex. This works combines the ideas of fiber photometry to access deep layers and using microprism to turn the …
Reviewer #2 (Public Review):
This manuscript reported a new approach to conduct neural activity imaging and manipulation in two different cortical layers. Two periscopes, each constructed from a micro-prism, a GRIN lens and a multi-mode fiber, could be inserted to the brain at different depths, and each can either perform imaging or optogenetics. The authors demonstrated a few applications: stimulation of L5 soma and superficial layer dendrites to evoke backpropagating action potential; optogenetically stimulating cells in L2/3 and observing response in L5 to investigate interaction between cells in two different layers; and simultaneously recording axon terminals from posteromedial thalamic nucleus at two different depths in cortex. This works combines the ideas of fiber photometry to access deep layers and using microprism to turn the optical field of view by 90 deg.
Major strengths:
• Using microprism to perform layer specific imaging or optogenetics.
• Low cost
• Demonstrations of a few applications that require layer specific imaging and optogenetics.Major weakness:
• As this is an inherently a variation of fiber photometry, there is a lack of cellular resolution and there is tissue damage.
• Innovation is modest, as it is an incremental improvement of fiber photometry. Some of the applications may be performed through regular fiber photometry as well.
• There is a lack of details on the optical setup and characterization of the periscope, i.e. how to choose the fiber, GRIN lens; optical throughput etc.Overall, this research provides a new method to image/manipulate the neural activity of two different cortical layers. However, more details are needed on the optical setup and characterization of the periscope. The innovation of this work is modest.
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