Mechanical stimulus delivery with the Automated Reproducible Mechanostimulator (ARM)

(A) Comparison between manual stimulus delivery that requires a researcher to aim and deliver stimulus by hand in close proximity to mice vs robotic stimulus delivery via the ARM using motorized linear stages to maneuver and deliver stimulus and a bottom camera using a superimposed calibrated crosshair to aim. (B) Zoomed-in schematic showing components of the ARM including the configuration of the linear axi, the holder attaching the aiming camera to the ARM, the stimulus holder, and the rotational axis that allows for switching between stimuli without detaching components or needing to enter the room. (C) An ARM vs manual stimulus aim comparison was conducted by 5 researchers who delivered 10 instances each of manual and ARM pinprick stimulus to a stationary target. A significant (p<0.0001) 93.3% decrease in distance off-target was observed in ARM stimuli delivery compared to manual delivery.

The ARM decreases variability in von Frey hair stimulus delivery.

(A) The ARM and external testers each first applied vFH stimulus to a force sensor (1.4g, 2g) before applying stimuli to a cohort of mice (n=10) and comparing behavior (0.02g, 0.07g, 0.16g, 0.6g, 1g, 1.4g). (B) Researchers and the ARM user were told to apply stimulus for 2 seconds to the force sensor. Time on the sensor was measured. (C) Standard deviation of all force sensor trials, normalized based on application start time. (D) Coefficient of variance for vFH (0.6g, 1g, 1.4g, 2g) on target time as determined by the force sensor was calculated for the ARM and compared to each researcher (p=0.0211), and the combined manual trials(p<0.0001) with a one-way anova. (E) Both researchers and the ARM tested a cohort of wildtype mice (n=10), applying each vFH 10 times to each mouse, producing the expected vFH response curves, includes SEM. (F) Each set of 10 vFH applications was timed for both manual and ARM stimulus delivery, with the ARM taking on average 50.9% less time to perform each set of applications (p<0.0001, 2-tailed paired t-test).

Updates to pain assessment at withdrawal speeds(PAWS) analysis.

(A) Schematic outlining high-speed recording to pose tracking (DLC or SLEAP) to updated PAWS software pipeline. The blue dotted line denotes beginning of withdrawal response and t* denotes the peak of the initial reflexive paw withdrawal response, with reflexive features including max height and max Y velocity measured pre t* and affective features including shaking and paw distance traveled measured post t*.(B) Stimulus flexible paw withdrawal latency measurement, made possible by syncing ARM stimulus with high-speed video recordings, separates between responses cotton swab and pinprick stimuli(p=0.0081). (C) Test of new PAWS pipeline using carrageenan inflammatory pain model. PAWS detected significantly higher number of paw shakes 4 hours after injection than baseline(p=0.0385).

Remote delivery of mechanical stimuli reveals the effects of researcher presence.

(A) Schematic showing the remote operation of the ARM allowing for researcher-agnostic experiments and flexibility. (B) Male mice (n=10) were habituated either with a researcher present or not for 3 days. Across the 3 days mice rested for the full minute significantly sooner than those with a researcher present (p=0.0217). (C) The number of times each mouse turned as measured during two 1-minute windows 20-30 minutes each day, normalized by each groups turning behavior during the first 10 minutes of day 1. On day 2 the remote-habituated mice showed significantly decreased turning behavior compared to those habituated with a researcher present (p=0.024). Only the remote-habituated mice showed significantly decreased turning behavior on day 3 compared to day 1 (p=0.0234). (D) Experimental schematic showing remote ARM stimulus delivery with either a researcher or no researcher in the room. (E-F) A 2-way Anova found significant differences in max paw height(p=0.0413) and max Y velocity (p=0.0406) in response to cotton swab for male mice when researcher 2 was present compared to no researcher. (G) Sex-dependent differences were found in response to cotton swab when Researcher 1 was present for distance traveled(p=0.0468). (H-J) Sex-dependent differences were found in response to pinprick stimuli when Researcher 2 was present, but not other conditions for max paw height(p=0.0436), max Y velocity(p=0.0424), and distance traveled (p=0.0038). Male mice showed significant differences in paw distance traveled(P=0.0149) when Researcher 2 was present compared to when none was.

Isolating the effect of variation in the application of pinprick stimulus.

(A) Schematic showing how stimulus delivery variation was modeled through changing pinprick intensity by increasing/decreasing pinprick apex and velocity. (B-C) Reflexive features were found to correlate with stimulus intensity based on a simple linear regression, withdrawal latency with a negative correlation and max paw height with a positive correlation. (D-E) For affective features, paw shaking time showed no significant correlation with stimulus intensity and paw distance traveled showed a positive correlation.

Linking ARM stimulation with behavior and cellular-resolved brain activity in the basolateral amygdala (BLA).

(A) Schematic showing alignment of BLA neural activity recorded by a microendoscope, PAWS behavioral features, and stimulus facilitated by the ARM. (B) Confirmation of injection of jGCaMP8f virus and insertion of Inscopix mini-scope to the BLA. (C-D) Cell map from processed mini-scope recording with a selection of representative deconvolved cell traces in pseudocolors over a 1000 sec window. (E-F) Example traces and cell map of pinprick stimulus aligned up and down-regulated cells based on peri-event analysis. (G) Results of peri-event analysis with up and down-regulated cells based on stimulus, and comparison with random background events. Total regulated cells increased compared to background control for all stimuli (p<0.0001). (H) Percentage of cells registered across multiple days that are regulated during response to mechanical touch and/or pain stimuli. (I) Pearson correlation between the fraction of total of peri-event analysis identified mechanical pain-regulated cells with matching regulation for each stimulus event with withdrawal latency (J) and distance traveled in the 1.5 seconds post-stimulus application.

ARM stimulus delivery and comparison with manual delivery.

(A) Schematic showing vFH wheel mounted on the ARM allowing for seamless switching between full range of vFH filaments and sin wave movement of ARM allowing for full application vFH max force for 2 sec. (B) ARM-based application of cotton swab and pinprick stimuli via sin wave motion mimicking manual delivery. (C) Comparison of pinprick stimuli delivered manually and via the ARM, based on max stimulus height measured via high-speed video recordings. Error rate of +/- 0.152 mm based on resolution.

Cross-researcher vFH behavior comparison manual versus ARM delivery.

(A) Comparison between paw withdrawal frequency elicited by Researcher 1 versus Researcher 2 with 2-way Anova. Significant differences were found in behavior elicited by 0.6g (p=0.0034), 1g (p=0.0462), and overall (p=0.0008). (B) Two researchers applied ARM vFH stimulus remotely over two days. 2-way Anova detected no significant differences.

Test of 500 fps PAWS analysis.

(A) Cohort of male mice (n=10) tested with cotton swab and pinprick stimuli. Pinprick was found to elicit significantly increased max paw height (p=0.0163), (B) max Y velocity (p=0.0034), and (C) paw distance traveled (p=0.0172) by a paired t-test. (D) Number of paw shakes was higher for pinprick stimuli but was not found to be significant.

Shaking duration was found to not significantly change based on experimenter presence.

(A-B) Remote experiment comparing mouse response when one of two researchers is present vs none. Non significant sex-dependent differences were found in response to cotton swab (p=0.0613) and pinprick(p=0.0551) when Researcher 1 was present for paw shaking duration.

Isolating the effect of variation in applying pinprick stimulus in female mice.

(A) Based on a simple linear regression withdrawal latency negatively correlates with stimulus intensity. (B) A piecewise linear regression analysis found that max paw height positively correlates with stimulus intensity for stim apex 1-3mm and negatively correlates for 3-4.5. (D-E) For affective features, paw shaking time and paw distance traveled showed no significant correlation with stimulus intensity.

Correlation of additional PAWS features with BLA mechanical pain neuron regulation.

(A) Example cell activity heat map and mean cell trace results of peri-event analysis of representative traces based on either 10 random background or pinprick events. (B) Example mean cell trace results of peri-event analysis of representative traces based on either 10 random background or pinprick events. (C) Fraction of peri-event analysis identified mechanical pain-regulated cells with matching regulation for each stimulus event. Cotton swab events showed decreased down(p<0.0001), up(p<0.05), and total(p<0.0005) regulation of identified mechanical pain cells compared to pinprick or max pinprick. (D) Pearson correlation between the fraction of total regulation of identified mechanical pain cells and paw max height and (E) max paw Y velocity.