Precision cutaneous stimulation in freely moving mice
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Curated by eLife
eLife Assessment
This valuable study combines real-time keypoint tracking with transdermal activation of sensory neurons to investigate sensory neuron recruitment in freely moving mice, and builds on the authors' prior work in stationary mice. The evidence supporting the utility of the system is solid, although a more thorough classification of the behavioral responses to nociceptor stimulation would strengthen the work. Importantly, future analyses could include other cutaneous sensory neuron subtypes, and could also be adapted for studying more complex behaviors. The work will be of interest to sensory biologists and pain researchers.
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Abstract
Somatosensation connects animals to their immediate environment, shaping critical behaviors essential for adaptation, learning, and survival. Probing the relationships between somatosensory inputs and behavior in mice presents substantial challenges, primarily due to the practical difficulties of delivering stimuli to the skin in moving mice. To address this problem, we have developed a system for precise cutaneous stimulation of mice as they walk and run through environments. The system employs real-time body part tracking and targeted optical stimuli, offering precision while preserving the naturalistic context of the behaviors studied to overcome the traditional trade-offs between precision and animal behavior. We demonstrate the system across nociceptive testing conducted in standard small chambers to behavior in large complex environments, such as mazes. We observed that cutaneous inputs evoke rapid responses, which modify behavior when stimuli are applied during motion. This system provides a means to explore the diverse and integrative nature of somatosensation, from reflexes to decision-making, in naturalistic settings.
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eLife Assessment
This valuable study combines real-time keypoint tracking with transdermal activation of sensory neurons to investigate sensory neuron recruitment in freely moving mice, and builds on the authors' prior work in stationary mice. The evidence supporting the utility of the system is solid, although a more thorough classification of the behavioral responses to nociceptor stimulation would strengthen the work. Importantly, future analyses could include other cutaneous sensory neuron subtypes, and could also be adapted for studying more complex behaviors. The work will be of interest to sensory biologists and pain researchers.
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Reviewer #1 (Public review):
Summary:
This study presents a system for delivering precisely controlled cutaneous stimuli to freely moving mice by coupling markerless real-time tracking to transdermal optogenetic stimulation, using the tracking signal to direct a laser via galvanometer mirrors. The principal claims are that the system achieves sub-mm targeting accuracy with a latency of <100 ms. The nature of mouse gait enables accurate targeting of forepaws even when mice are moving.
Strengths:
The study is of high quality and the evidence for the claims is convincing. There is increasing focus in neurobiology in studying neural function in freely moving animals, engaged in natural behaviour. However, a substantial challenge is how to deliver controlled stimuli to sense organs under such conditions. The system presented here constitutes …
Reviewer #1 (Public review):
Summary:
This study presents a system for delivering precisely controlled cutaneous stimuli to freely moving mice by coupling markerless real-time tracking to transdermal optogenetic stimulation, using the tracking signal to direct a laser via galvanometer mirrors. The principal claims are that the system achieves sub-mm targeting accuracy with a latency of <100 ms. The nature of mouse gait enables accurate targeting of forepaws even when mice are moving.
Strengths:
The study is of high quality and the evidence for the claims is convincing. There is increasing focus in neurobiology in studying neural function in freely moving animals, engaged in natural behaviour. However, a substantial challenge is how to deliver controlled stimuli to sense organs under such conditions. The system presented here constitutes notable progress towards such experiments in the somatosensory system and is, in my view, a highly significant development that will be of interest to a broad readership.
Weaknesses:
(1) "laser spot size was set to 2.00 } 0.08 mm2 diameter (coefficient of variation = 3.85)" is unclear. Is the 0.08 SD or SEM? (not stated). Also, is this systematic variation across the arena (or something else)? Readers will want to know how much the spot size varies across the arena - ie SD. CV=4 implies that SD~7 mm. ie non-trivial variation in spot size, implying substantial differences in power delivery (and hence stimulus intensity) when the mouse is in different locations. If I misunderstood, perhaps this helps the authors to clarify. Similarly, it would be informative to have mean & SD (or mean & CV) for power and power density. In future refinements of the system, would it be possible/useful to vary laser power according to arena location?
(2) "The video resolution (1920 x 1200) required a processing time higher than the frame interval (33.33 ms), resulting in real-time pose estimation on a sub-sample of all frames recorded". Given this, how was it possible to achieve 84 ms latency? An important issue for closed-loop research will relate to such delays. Therefore please explain in more depth and (in Discussion) comment on how the latency of the current system might be improved/generalised. For example, although the current system works well for paws it would seem to be less suited to body parts such as the snout that do not naturally have a stationary period during the gait cycle.
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Reviewer #2 (Public review):
Parkes et al. combined real-time keypoint tracking with transdermal activation of sensory neurons to examine the effects of recruitment of sensory neurons in freely moving mice. This builds on the authors' previous investigations involving transdermal stimulation of sensory neurons in stationary mice. They illustrate multiple scenarios in which their engineering improvements enable more sophisticated behavioral assessments, including (1) stimulation of animals in multiple states in large arenas, (2) multi-animal nociceptive behavior screening through thermal and optogenetic activation, and (3) stimulation of animals running through maze corridors. Overall, the experiments and the methodology, in particular, are written clearly. However, there are multiple concerns and opportunities to fully describe their …
Reviewer #2 (Public review):
Parkes et al. combined real-time keypoint tracking with transdermal activation of sensory neurons to examine the effects of recruitment of sensory neurons in freely moving mice. This builds on the authors' previous investigations involving transdermal stimulation of sensory neurons in stationary mice. They illustrate multiple scenarios in which their engineering improvements enable more sophisticated behavioral assessments, including (1) stimulation of animals in multiple states in large arenas, (2) multi-animal nociceptive behavior screening through thermal and optogenetic activation, and (3) stimulation of animals running through maze corridors. Overall, the experiments and the methodology, in particular, are written clearly. However, there are multiple concerns and opportunities to fully describe their newfound capabilities that, if addressed, would make it more likely for the community to adopt this methodology:
The characterization of laser spot size and power density is reported as a coefficient of variation, in which a value of ~3 is interpreted as uniform. My interpretation would differ - data spread so that the standard deviation is three times larger than the mean indicates there is substantial variability in the data. The 2D polynomial fit is shown in Figure 2 - Figure Supplement 1A and, if the fit is good, this does support the uniformity claim (range of spot size is 1.97 to 2.08 mm2 and range of power densities is 66.60 to 73.80 mW). The inclusion of the raw data for these measurements and an estimate of the goodness of fit to the polynomials would better help the reader evaluate whether these parameters are uniform across space and how stable the power density is across repeated stimulations of the same location. Even more helpful would be an estimate of whether the variation in the power density is expected to meaningfully affect the responses of ChR2-expressing sensory neurons.
While the error between the keypoint and laser spot error was reported as ~0.7 to 0.8 mm MAE in Figure 2L, in the methods, the authors report that there is an additional error between predicted keypoints and ground-truth labeling of 1.36 mm MAE during real-time tracking. This suggests that the overall error is not submillimeter, as claimed by the authors, but rather on the order of 1.5 - 2.5 mm, which is considerable given the width of a hind paw is ~5-6 mm and fore paws are even smaller. In my opinion, the claim for submillimeter precision should be softened and the authors should consider that the area of the paw stimulated may differ from trial to trial if, for example, the error is substantial enough that the spot overlaps with the edge of the paw.
As the major advance of this paper is the ability to stimulate animals during ongoing movement, it seems that the Figure 3 experiment misses an opportunity to evaluate state-dependent whole-body reactions to nociceptor activation. How does the behavioral response relate to the animal's activity just prior to stimulation?
Given the characterization of full-body responses to activation of TrpV1 sensory neurons in Figure 4 and in the authors' previous work, stimulation of TrpV1 sensory neurons has surprisingly subtle effects as the mice run through the alternating T maze. The authors indicate that the mice are moving quickly and thus that precise targeting is required, but no evidence is shared about the precision of targeting in this context beyond images of four trials. From the characterization in Figure 2, at max speed (reported at 241 +/- 53 mm/s, which is faster than the high speeds in Figure 2), successful targeting occurs less than 50% of the time. Is the initial characterization consistent with the accuracy in this context? To what extent does inaccuracy in targeting contribute to the subtlety of affecting trajectory coherence and speed? Is there a relationship between animal speed and disruption of the trajectory?
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Reviewer #3 (Public review):
Summary:
To explore the diverse nature of somatosensation, Parkes et al. established and characterized a system for precise cutaneous stimulation of mice as they walk and run in naturalistic settings. This paper provides a framework for real-time body part tracking and targeted optical stimuli with high precision, ensuring reliable and consistent cutaneous stimulation. It can be adapted in somatosensation labs as a general technique to explore somatosensory stimulation and its impact on behavior, enabling rigorous investigation of behaviors that were previously difficult or impossible to study.
Strengths:
The authors characterized the closed-loop system to ensure that it is optically precise and can precisely target moving mice. The integration of accurate and consistent optogenetic stimulation of the …
Reviewer #3 (Public review):
Summary:
To explore the diverse nature of somatosensation, Parkes et al. established and characterized a system for precise cutaneous stimulation of mice as they walk and run in naturalistic settings. This paper provides a framework for real-time body part tracking and targeted optical stimuli with high precision, ensuring reliable and consistent cutaneous stimulation. It can be adapted in somatosensation labs as a general technique to explore somatosensory stimulation and its impact on behavior, enabling rigorous investigation of behaviors that were previously difficult or impossible to study.
Strengths:
The authors characterized the closed-loop system to ensure that it is optically precise and can precisely target moving mice. The integration of accurate and consistent optogenetic stimulation of the cutaneous afferents allows systematic investigation of somatosensory subtypes during a variety of naturalistic behaviors. Although this study focused on nociceptors innervating the skin (Trpv1::ChR2 animals), this setup can be extended to other cutaneous sensory neuron subtypes, such as low-threshold mechanoreceptors and pruriceptors. This system can also be adapted for studying more complex behaviors, such as the maze assay and goal-directed movements.
Weaknesses:
Although the paper has strengths, its weakness is that some behavioral outputs could be analyzed in more detail to reveal different types of responses to painful cutaneous stimuli. For example, paw withdrawals were detected after optogenetically stimulating the paw (Figures 3E and 3F). Animals exhibit different types of responses to painful stimuli on the hind paw in standard pain assays, such as paw lifting, biting, and flicking, each indicating a different level of pain. Improving the behavioral readouts from body part tracking would greatly strengthen this system by providing deeper insights into the role of somatosensation in naturalistic behaviors. Additionally, if the laser spot size could be reduced to a diameter of 2 mm², it would allow the activation of a smaller number of cutaneous afferents, or even a single one, across different skin types in the paw, such as glabrous or hairy skin.
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