Mapping the neuroethological signatures of pain, analgesia, and recovery in mice

Abstract

Ongoing pain is driven by the activation and modulation of pain-sensing neurons, affecting physiology, motor function, and motivation to engage in certain behaviors. The complexity of the pain state has evaded a comprehensive definition, especially in non-verbal animals. Here, in mice, we used site-specific electrophysiology to define key time points corresponding to peripheral sensitivity in acute paw inflammation and chronic knee pain models. Using supervised and unsupervised machine learning tools, we uncovered sensory-evoked coping postures unique to each model. Through 3D pose analytics, we identified movement sequences that robustly represent different pain states and found that commonly used analgesics do not return an animal's behavior to a pre-injury state. Instead, these analgesics induce a novel set of spontaneous behaviors that are maintained even after resolution of evoked pain behaviors. Together, these findings reveal previously unidentified neuroethological signatures of pain and analgesia at heightened pain states and during recovery.

Keywords: analgesia; behavior; computer vision; electrophysiology; machine learning; motion sequencing; mouse; nociception; pain; recovery.

Figure 1.. Carrageenan and MIA pain models cause changes in ethological and evoked behavior accompanied by alterations in sensory neuron excitability over distinct timescales.

Timeline of experiments following (A) unilateral intraplantar injection of carrageenan or (B) intra-articular injection of MIA. (C) Inflammation of the injected (Ipsi) hind paw was observed 4-hours post-injection compared to the non-injected (Contra) paw. (D) Footpad swelling was quantified with digital calipers following injection of carrageenan. (E) Histological examination of knee joints 10 days after injection showed a healthy layer of cartilage for the contralateral joint (Ei), which had been lost in the ipsilateral joint (Eii). (F) Knee joint swelling was measured following injection of MIA. (G) The time mice spent rearing before, 4-hours, and 24-hours post-induction of inflammation with carrageenan was assessed using a dynamic weight bearing device. (H) Hargreaves measurement of carrageenan-induced heat hypersensitivity was assessed at baseline and following 4- and 24-hours. (I) Time spent rearing was assessed at baseline (BSL) and 3- (D 3) and 10-days (D 10) post-injection of MIA. (J) Sensitivity of both knee joints to mechanical stimulation was tested using a pressure application measurement device before and 3- and 10-days post-injection of MIA. (K) Schematic representation of retrograde labeling of hind paw innervating sensory neurons with Fast Blue followed by cell culture and whole cell patch clamp electrophysiology. (L) Representative current clamp recordings of Ipsi and Contra hind paw neurons of comparable capacitance, showing action potentials evoked by ramp injection of current (0–1 nA, 1 s), the thresholds for action potential discharge are annotated with dashed (Contra) or solid (Ipsi) lines. (M) Schematic representation of retrograde labeling of knee innervating sensory neurons with Fast Blue followed by cell culture and whole cell patch clamp electrophysiology. (N) Representative current clamp recordings of Ipsi and Contra knee neurons of comparable capacitance, showing action potentials evoked by ramp injection of current (0–1 nA, 1 s), the thresholds for action potential discharge are annotated with dashed (Contra) or solid (Ipsi) lines. (O) Step-wise current injections were used to determine the rheobase of Ipsi and Contra hind paw innervating sensory neurons 4- or 24-hours post-induction of inflammation with carrageenan. (P) Neurons with rheobase < 450 pA were stimulated with a suprathreshold (2 x rheobase) for 500 ms and the number of action potentials discharged counted. (O) Step-wise current injections were used to determine the rheobase of Ipsi and Contra knee innervating sensory neurons 3- or 10-days post-injection of MIA. (P) Neurons with rheobase < 450 pA were stimulated with a suprathreshold (2 x rheobase) for 500 ms and number of action potentials discharged counted. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001: (C, E, G, H, I, J) one-way ANOVA with Bonferroni post hoc; (O, Q, R) Mann-Whitney test between Ipsi and Contra for individual time points.

Figure 2.. PAWS and B-SOiD automated pain assessment platforms detect defensive coping behaviors associated with pain sensation during inflammation.

(Ai) A behavioral response to a somatosensory stimulus at baseline. (Aii) Post-carrageenan injection mice guard the paw in the air for extended time. (i,ii) Green lines show paw trajectory pattern across entire behavior, and mouse image shows single frame with paw at its apex. (B) PAWS software measures reflexive (i.e. height, paw displacement along the y axis, etc.) and affective behavioral features (i.e. shaking, guarding, distance traveled by the paw, etc.). The apex t* or first peak of the behavioral response separates reflexive and affective behavioral features (as described in the methods from Jones et al., 2020). Here, the y axis is a univariate projection of the paw displacement across both x and y dimensions, in centimeters. This graph thus captures paw movements in both x and y directions over time, following dynamic brush stimulation at baseline, for 1 mouse. Further details on the computation methods used to generate this graph can be found in Jones et al., 2020. (C,D) Affective features such as paw guarding and paw shaking are upregulated in response to dynamic brush and light pinprick comparing baseline to 4- and 24-hours post-carrageenan injection. (E,F) Paw guarding was found upregulated at 10-days after MIA knee injection, consistent with the paw being a potential secondary site of hypersensitivity. (G) Low-dimensional projection of feature clusters as identified after UMAP/HDBSCAN. 5 colors were then assigned to the 11 identified sub-clusters to indicate their post-hoc behavioral group assignment. Stacked bar plots of the percent of time spent doing each behavior (rest, paw lift, angled guard, flat guard, hovering) in response to dynamic brush (H) and (I) light pinprick at baseline, 4-hours, and 24-hours time points post-carrageenan injection. Responses are color-coded by the identified action type as in panel (G). Examples of the angled paw guard identified by B-SOiD, which may be indicative of the activation of different subsets of sensory neurons (mechanoreceptors by brush, inducing (J) angled guard, nociceptors by pinprick, inducing (K) flat guard). N=10 mice per group; * p < 0.05, ** p < 0.01: Kruskal-Wallis test followed by Dunn’s multiple comparisons were performed to determine statistical significance between the responses of mice to each stimuli across time independently.

Figure 3.. 3D pose analysis detects behavioral signatures of paw carrageenan-induced inflammatory pain and knee MIA-induced injury.

(A) Schematic of analysis pipeline. (B) Linear discriminant analysis (LDA) of spontaneous behavior module usage at baseline, and following carrageenan injection at 4- and 24-hours. (C) LDA of spontaneous behavior module usage at baseline, and 3- and 10-days post MIA injection. Spinogram representations of micro-movements that define particular behavioral modules identified as (D) rearing, (E) pausing and (F) grooming. Usage of rearing decreases following induction of inflammation with carrageenan (G) and MIA (J). Usage of pausing (H, K) and grooming (I, L) increase following injection of carrageenan in the paw and MIA in the knee respectively. Baseline (BSL) n= 10 animals, 4h n= 20 animals, 24h n= 10 animals, D3 n= 20 animals, D10 n= 10 animals, Statistical analysis: corrected bootstrap t-test.

Figure 4.. Meloxicam relieves affective features of hyperalgesia but it does not promote return to pre-inflammation spontaneous behavior, while gabapentin improves spontaneous signatures of MIA-induced knee injury.

(A) Timeline of the experiment. Mice were tested at baseline and after intraplantar injection of 20 μl 3% carrageenan at 4-hours post-injection, then at 24-hours after intraperitoneal injection of saline or meloxicam. (B) Timeline of the experiment. Mice are tested at baseline and after intra articular knee injection of 10 μl 0.1mg/ul MIA at 3-days post-injection, then at 10-days after intraperitoneal injection of saline or gabapentin. (C) Hargreaves measurement of carrageenan-induced heat hypersensitivity at baseline, 24-hours following carrageenan injection, as well as pain relief by meloxicam at 24-hours, ipsi- (full circles) and contralateral (empty circles) to paw injection. (D) Measurement of MIA-induced pressure knee hypersensitivity at baseline, 10-days following MIA knee injection, as well as pain relief by gabapentin at 10-days, ipsi- (full squares) and contralateral (empty squares) to knee injection. (E) Paw guarding duration is measured with machine learning at baseline, and post-carrageenan injection at 4-hours, and 24-hours after saline or meloxicam intraperitoneal injection following dynamic brush (left) or light pinprick (right). (F) Paw guarding duration is measured with machine learning at baseline, and post-MIA knee injection at 3-days, and 10-days after saline or gabapentin intraperitoneal injection following dynamic brush (left) or light pinprick (right). (G-I) 3D pose analysis of spontaneous behavior of 5 groups: baseline + meloxicam intraperitoneal injection, baseline + saline intraperitoneal injection, 4-hours post-carrageenan paw injection, 24-hours post-carrageenan paw injection + saline intraperitoneal injection, 24-hours post-carrageenan paw injection + meloxicam intraperitoneal injection. (G) Spontaneous rearing behavior is decreased after paw carrageenan injection, further decreased by meloxicam intraperitoneal injection (example module #15 among other rearing modules downregulated, see Table 2). (H) Spontaneous grooming behavior is increased after paw carrageenan injection, further increased by meloxicam intraperitoneal injection (example module #60). (I) Representation of LDA of raw usage for the five different groups. (J-L) 3D pose analysis of spontaneous behavior 5 groups: baseline + gabapentin intraperitoneal injection, baseline + saline intraperitoneal injection, 3-days post-MIA knee injection, 10d post-MIA knee injection + saline intraperitoneal injection, 10-days post-MIA knee injection + gabapentin intraperitoneal injection. (J) Spontaneous rearing behavior is decreased after knee MIA injection, which can be resolved after gabapentin intraperitoneal injection (example module #18). (K) Locomotion is affected after knee MIA injection, which can be resolved after gabapentin intraperitoneal injection (example module #57). (L) Representation of LDA of raw usage for the five different groups. (I-L) While the point clouds in Figure 4I and 4L do show some overlap between conditions, cohorts can be distinguished by their module usage, which we quantified by computing the F1 of the LDA in predicting the condition of the held-out animals (F1-scores: CAR bsl+saline = 0.57, CAR bsl+meloxicam = 0.50, CAR 4h = 0.67, CAR 24h+saline = 0, CAR 24h+meloxicam = 0.40, overall model accuracy = 0.50 better than “pure-chance” = 0.20 and randomized data). For accuracy, we report F1, the harmonic mean of precision and recall. For 3D pose analysis, baseline+saline (bsl+sal) n= 10 animals, baseline+meloxicam (bsl+mel) n= 10 animals, baseline+gabapentin (bsl+gbp) n= 10 animals, 4h post-carrageenan n= 20 animals, 24h post-carrageenan+saline (24h+sal) n= 10 animals, 24h post-carrageenan+meloxicam (24h+mel) n= 10 animals, D3 post-MIA n= 20 animals, D10 post-MIA+saline (D10+sal) n= 10 animals, D10 post-MIA+gabapentin (D10+gbp) n= 10 animals, Statistical analysis: corrected bootstrap t-test.

Figure 5.. Higher order behavioral sequences predict pain and analgesic states in rodents.

(A-D) Example of module sequences most representative of spontaneous behavior at baseline (A), 4-hours following carrageenan paw injection (B), and 10-days post MIA knee injection and saline (C) or gabapentin (D) intraperitoneal injection, as identified by learned embeddings method. A standard co-location algorithm was first used to detect 2-long module sequences according to whether the 2-long sequence (e.g. A>B) appeared significantly more than each of its constituents (i.e. A or B). Wherever a significant 2-long sequence was detected, we replaced it by a new agglomerated syllable representing the co-location. We then recursed on this procedure to find 3- and 4-long sequences, at each iteration checking for the significance of agglomerated sequences by comparing their frequency to those of each of the sequence’s constituents. (E) Bar plot showing the relative performances of the different representations along with the performance of targeted (Targ. Abl.) vs random ablations (Rand. Abl.) for carrageenan dataset. (F) Bar plot showing the relative performances of the different representations along with the performance of targeted (Targ. Abl.) vs random ablations (Rand. Abl.) for MIA dataset. Baseline+saline (bsl+sal) n= 10 animals, baseline+meloxicam (bsl+mel) n= 10 animals, baseline+gabapentin (bsl+gbp) n= 10 animals, 4h post-carrageenan n= 20 animals, 24h post-carrageenan+saline (24h+sal) n= 10 animals, 24h post-carrageenan+meloxicam (24h+mel) n= 10 animals, D3 post-MIA n= 20 animals, D10 post-MIA+saline (D10+sal) n= 10 animals, D10 post-MIA+gabapentin (D10+gbp) n= 10 animals, Statistical analysis: corrected bootstrap t-test.

Figure 6.. 3D pose analysis resolves the behavior of animals following resolution of inflammation as a new state as opposed to return to baseline, this state is stabilized by treatment with the anti-inflammatory Meloxicam.

(A) Timeline of extended behavioral characterization following induction of inflammation with carrageenan. (B) Heat hypersensitivity of the Ipsi paw is comparable to baseline by 6-days post-injection of carrageenan. (C) Intraplantar injection of saline does not affect mechanical sensitivity of the hind paw when assessed with von Frey, however, hypersensitivity is seen at 1- and 3-days post injection of carrageenan which resolves by day 6. (D) Mutation plot summarizing how usage of each behavioral module identified via MoSeq changes with time following intraplantar injection of carrageenan. Usage of certain behaviors including (E) rearing and (F) grooming appear to recover with time following injection of carrageenan, although others including (G) rearing and (H) pausing remain different to baseline at 14-days post-injection. (I) Administration of a single dose of Meloxicam at 24-hours post-injection of carrageenan stabilizes spontaneous behaviors at time points where evoked-sensitivity has recovered. (J) Spontaneous behavior is more variable when tested after absence of pain relief. For 3D pose analysis, baseline+saline (bsl+sal) n= 10 animals, 4h post-carrageenan n= 20 animals, 24h post-carrageenan+saline (24h+sal) n= 10 animals, 6d post-carrageenan n= 20 animals, 14d post-carrageenan n= 20 animals, Statistical analysis: corrected bootstrap t-test.