Remote automated delivery of mechanical stimuli coupled to brain recordings in behaving mice

Abstract

The canonical framework for testing pain and mechanical sensitivity in rodents is manual delivery of stimuli to the paw. However, this approach is time consuming, produces variability in results, requires significant training, and is ergonomically unfavorable to the experimenter. To circumvent limitations in manual delivery of stimuli, we have created a device called the ARM (Automated Reproducible Mechano-stimulator). Built using a series of linear stages, cameras, and stimulus holders, the ARM is more accurate at hitting the desired target, delivers stimuli faster, and decreases variability in delivery of von Frey hair filaments. We demonstrate that the ARM can be combined with traditional measurements of pain behavior and automated machine-learning based pipelines. Importantly, the ARM enables remote testing of mice with experimenters outside the testing room. Using remote testing, we found that mice habituated more quickly when an experimenter was not present and experimenter presence leads to significant sex-dependent differences in paw withdrawal and pain associated behaviors. Lastly, to demonstrate the utility of the ARM for neural circuit dissection of pain mechanisms, we combined the ARM with cellular-resolved microendoscopy in the amygdala, linking stimulus, behavior, and brain activity of amygdala neurons that encode negative pain states. Taken together, the ARM improves speed, accuracy, and robustness of mechanical pain assays and can be combined with automated pain detection systems and brain recordings to map central control of pain.

Figure 1:. 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 to aim. (B) Zoomed-in schematic showing components of the ARM including the configuration of the linear axi, the aiming camera to the ARM, and the stimulus holder. (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.

Figure 2:. 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 for 1.4g (C) and 2g vFH’s. (D) Stimulus delivery time for 1.4g and 2g force sensor trial. (E) 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. (F) 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. (G) 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). (H) Two researchers applied ARM vFH stimulus remotely over two days. 2-way Anova detected no significant differences.

Figure 3:. ARM System and integration with pain assessment at withdrawal speeds(PAWS) analysis.

(A) Groups of mice will be tested using the ARM, (B) and for each stimulus delivery reflexive features including withdrawal latency and mechanical threshold are measured automatically using a force sensor incorporated into the device. (C) Pose analysis of the integrated 500 fps cameras and PAWS high-speed analysis then measures the extent of the reaction including max paw height, velocity, and behaviors associated with the affective aspect of pain including distance traveled, shaking and guarding. (D) Integration with load cell allows for customizable force ramp stim, where force starts low and ramps up over time, and (E) a consistent stimulus that hold at a set force and retracts after duration exceeded or paw withdrawal. (F) Test of new PAWS pipeline using carrageenan inflammatory pain model, detected significantly higher number of paw shakes at 4 hours compared to baseline (p=0.039), and (G) decreased mechanical threshold with the initial version of the force ramp stimulus (p=0.027). (H) An updated version of the force stimulus was used with a CFA model and found significant decreases compared to control and baseline (p=0.044, 0.025) with the ramp stimulus and a highly significant decrease (p<0.0001) in withdrawal latency in response to a 1 g stimulus.

Figure 4:. 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. (K) To test whether the gender of the experimenter present effects behavior cohorts of male and female mice (N=15) were tested across 3 weeks with groups of male and female researchers (N=3) and no researcher controls. Male researchers induced significantly decreased mechanical threshold in female vs male mice (p=0.034).

Figure 5:. 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.

Figure 6:. 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.