Scanned optogenetic control of mammalian somatosensory input to map input-specific behavioral outputs

  1. Ara Schorscher-Petcu
  2. Flóra Takács
  3. Liam E Browne

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Abstract

Somatosensory stimuli guide and shape behavior, from immediate protective reflexes to longer-term learning and higher-order processes related to pain and touch. However, somatosensory inputs are challenging to control in awake mammals due to the diversity and nature of contact stimuli. Application of cutaneous stimuli is currently limited to relatively imprecise methods as well as subjective behavioral measures. The strategy we present here overcomes these difficulties, achieving ‘remote touch’ with spatiotemporally precise and dynamic optogenetic stimulation by projecting light to a small defined area of skin. We mapped behavioral responses in freely behaving mice with specific nociceptor and low-threshold mechanoreceptor inputs. In nociceptors, sparse recruitment of single-action potentials shapes rapid protective pain-related behaviors, including coordinated head orientation and body repositioning that depend on the initial body pose. In contrast, activation of low-threshold mechanoreceptors elicited slow-onset behaviors and more subtle whole-body behaviors. The strategy can be used to define specific behavioral repertoires, examine the timing and nature of reflexes, and dissect sensory, motor, cognitive, and motivational processes guiding behavior.

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  1. Version published to 10.7554/elife.62026 on eLife
  2. eLife

    ###Reviewer #3:

    In this manuscript, Schorscher-Petcu et al., describe a very exciting new approach combining precise optogenetic stimulation of cutaneous nerve terminals with high-speed imaging for machine-guided behavior analysis. This work is timely, and there are many clear applications to understand peripheral somatosensory encoding using this strategy. More thorough methodology and guidance for future end users could be provided. However, I am much less enthusiastic about the conclusions drawn for a sparse neural coding hypothesis, based on the data presented. Significant support for this hypothesis would require more substantial revisions, including testing in mouse lines to target other specific sensory modalities, innervation regions, and possibly pain states.

    Substantive concerns:

    1. A major strength would be the ability to combine precise optogenetic stimulation with other behavioral assays. Can this be used in combination with existing nociceptive tests? For example, does the NIR-FTIR allow for tracking of spontaneous pain behaviors after intraplantar formalin or CFA? And can this then also be used to assess sensitization of genetically-identified fibers using scanned optogenetics?

    2. What is the rationale for varying the pulse-widths rather than light intensity for these experiments? Increasing light intensity will generally lead to larger ChR2 photocurrents, while changing light duration generally affects deactivation and desensitization kinetics. At a peripheral terminal, the effects of subthreshold depolarization may in fact mimic the physiological activation of endogenous receptors, like TRP channels. This level of fine-tuned control would be a significant advancement for understanding how information from different somatosensory modalities is processed and integrated.

    3. It would be useful to have more thorough characterization of the strengths and limitations of the optical system. For example, how quickly are the spatially patterned stimuli able to be moved? What is the maximal area for a single spot or array of spots, and how long does this take to scan? Does the time between patterned stimuli, both in a single spot or when spatially distributed, alter withdrawal responses? How quickly can the beam spot size be altered? These will be important points that potential users will need to consider before building this system.

    4. It would also be extremely helpful to provide more thorough details and discussion of implementing Deep Lab Cut analysis with this system.

    5. The proposed activation of myelinated A fibers is very surprising given the opsin expression patterns in TRPV1:ChR2 mice. The authors cite Arcourt et al., however they did not find any expression of TRPV1 in their genetically-defined A-fiber nociceptors. And with this breeding strategy can the authors please clarify and provide support for this apparent discrepancy?

    6. The response latencies in Figure 3 fit well with the hypothesis that fibers with different conduction velocities are activated by changing pulse areas. Do different stimulus intensities (or durations) preferentially activate A vs C-fiber afferents akin to electrical stimulation of dorsal roots in spinal cord recordings? Or does the larger stimulation area merely increase the probability that an A nerve ending is in the illuminated region? Could this alternatively be explained by additive depolarization or more complex spike interference at these axon collaterals that branch extensively in the skin? Also, do the response profiles vary after activation of a presumptive A vs C-fiber?

    7. Is the pain-related behavior in response to single or patterned optogenetic stimulation reduced by analgesics acting centrally or peripherally? This could reveal important differences in rapid reflex or protective behaviors and more complicated nocifensive responses, and support the author's claims of true pain-related behaviors.

  3. eLife

    ###Reviewer #2:

    The manuscript by Schorscher-Petcu et al developed a method/system for scanned optogenetic activation of nociceptors on the paw in freely behaving TrpV1-Cre::ChR2 mice, with concurrent measure of both paw responses (using near-infrared frustrated total internal reflection to measure paw/floor contacts) and full body responses (scoured using DeepLabCut). Using this approach, they showed that the number of activated nociceptors governs the timing and magnitude of rapid protective pain-related behavior. The detailed description of how to construct the setup, and the open availability of the software are useful for other labs to apply this method.

    I have three points that I would like the authors to address:

    1. I have a hard time evaluating the hierarchical bootstrap procedure, which references a pre-print. Is this method really ensuring that the results are more rigorous? Or is it needlessly complicating the reporting of fairly simple metrics for what appear to be obvious phenomena (Figure 3) like paw rise time?

    2. I have an issue with the word "sparse code". In neuroscience in general, sparse code refers to the phenomenon that a given stimulus only activates a very small percentage of neurons in a population. Here the authors refer to a single action potential elicited by optogenetic stimulus. Some other term should be used.

    3. For Figure 4 (whole body movement), the analysis should be using a vector instead of a scalar. The example in Figure 4D clearly shows directionality, i.e. the nose moves toward the stimulated paw. But the authors only analyzed maximum distance (a scaler, not vector). So the correlation here in Figure 4F is showing "when body part A moves a lot, does body part B also move a lot". Instead, I think the analysis more in line with the examples would be when body part A moves one direction, the direction of movement of body part B would be correlated. In other words, the analysis needs to be done where distance is some kind of vector, either closer to or further away from the paw or moving toward or away from the stimulated paw.

  4. eLife

    ###Reviewer #1:

    The manuscript by Schorscher-Petcu is a very innovative study addressing an important problem in pain and somatosensory neuroscience - precise and remote delivery of sensory stimuli. The strength of this work is the experimental paradigm, as the biological insight seems quite weak and not more expansive than previous work from the authors and others in the field. One has to ask, is this work being sold on the tool or new biology? If it were the latter, this work could easily benefit by comparing the data with Trpv1-ChR2 with other sensory neuron populations - as the authors mention in the discussion. Nonetheless, the rationale for such a tool developed here is widely agreed upon in the field, and if others can easily adopt this strategy, this could become the standard for peripheral optogenetic stimulation of the hind paw.

    Major comments:

    1. It remains unclear to me how one actually remotely aims at the hind paw of interest. Is there a joystick where one aims at the paw? Relatedly, are there ever any misfires where one intends to aim at the paw but hits another area? Or does the mouse sometimes move when you intend to hit one area thus causing an unintended stimulus delivery?

    2. In Figure 2 the authors cite their previous studies which demonstrate that a brief optogenetic stimulus to the paw elicits a single action potential which is capable of causing a behavioral response. The authors then infer here that their nanosecond manipulation of light also influences single action potentials. However, without verifying that in this new experimental context, simply citing the older work is insufficient evidence to draw any correlation to action potentials.

    3. In Figure 3 the authors mention that in a fraction of trials (presumably ~35%) the paw moved but did not withdraw, and that this was detected by the acquisition system and not by eye. I am confused about what the authors are considering a paw withdrawal. Is not any paw lift also a withdrawal? Additionally, how can the acquisition system see things that cannot be seen by the experimenter? Could this point towards an error of the system? Is there an independent validation of how well the system is working compared to some benchmark?

  5. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.

    ###Summary:

    The manuscript by Schorscher-Petcu is a very innovative study approaching an important problem in pain and somatosensory neuroscience - precise and remote delivery of sensory stimuli. This work is timely, and there are many clear applications to understanding peripheral somatosensory encoding using this strategy. The rationale for such a tool developed here is widely agreed upon in the field, and if others can easily adopt this strategy, this could become the standard for peripheral optogenetic stimulation of the hind paw.

  6. Version published to 10.1101/2020.08.10.244046 on bioRxiv