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. 2020 Mar 17;7(2):ENEURO.0210-19.2020.
doi: 10.1523/ENEURO.0210-19.2020. Print Mar/Apr 2020.

The Operant Plantar Thermal Assay: A Novel Device for Assessing Thermal Pain Tolerance in Mice

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Free PMC article

The Operant Plantar Thermal Assay: A Novel Device for Assessing Thermal Pain Tolerance in Mice

Ashlie N Reker et al. eNeuro. .
Free PMC article

Abstract

Pain is a multidimensional experience of sensory-discriminative, cognitive, and affective processes; however, current basic research methods rely heavily on response to threshold stimuli, bypassing the supraspinal processing that ultimately gives rise to the pain experience. We developed the operant plantar thermal assay (OPTA), which utilizes a novel, conflict-based operant task requiring evaluation and active decision-making to obtain reward under thermally aversive conditions to quantify thermal pain tolerance. In baseline measures, male and female mice exhibited similar temperature preferences, however in the OPTA, female mice exhibited greater temperature-dependent tolerance, as defined by choice time spent in an adverse thermal condition to obtain reward. Increasing reward salience (4% vs 10% sucrose solution) led to increased thermal tolerance for males but not females. To determine whether neuropathic and inflammatory pain models alter thermal tolerance, animals with chronic constriction injury (CCI) or complete Freund's adjuvant (CFA), respectively, were tested in the OPTA. Surprisingly, neuropathic animals exhibited increased thermal tolerance, as shown by greater time spent in the reward zone in an adverse thermal condition, compared with sham animals. There was no effect of inflammation on thermal tolerance. Administration of clonidine in the CCI model led to increased thermal tolerance in both injured and sham animals. In contrast, the non-steroidal anti-inflammatory meloxicam was anti-hyperalgesic in the CFA model, but reduced thermal pain tolerance. These data support the feasibility of using the OPTA to assess thermal pain tolerance to gain new insights into complex pain behaviors and to investigate novel aspects of analgesic efficacy.

Keywords: analgesia; inflammatory pain; neuropathic pain; novel methods; operant learning.

Figures

Figure 1.
Figure 1.
OPTA apparatus and animal tracking. A, Schematic of the OPTA. Each power supply runs both a Peltier and a temperature controller. The temperature controller receives feedback from a thermode attached to the aluminum plate, allowing independent real-time thermal control. The acrylic enclosure creates a choice paradigm by isolating each floor into chambers between which a narrow pass exists. The video system tracks the head of the animal. B, A representative heatmap illustrating the approach-avoidance conflict. The majority of activity is seen on the null (30°C) side, with increased activity apparent at pass point between chambers, while activity in the aversive reward side is largely limited to the reward zone. C, Photograph of the OPTA in operation.
Figure 2.
Figure 2.
Thermal preference of adult naive wild-type mice, no reward. Naive male (n = 5) and female (n = 5) mice were monitored for 900 s (600 s for 35/40°C) on pseudo-randomly presented paired-temperature preference tests. Ratio of time spent in each chamber per test is shown. No between sex differences were detected. Paired t test (two-tailed). Data are presented as mean ± SEM, *p ≤ 0.05, **p ≤ 0.01.
Figure 3.
Figure 3.
Training for operant acquisition. A, Female (n =8) and male (n =8) mice showed no difference in time spent in reward zone across 4 d of training, one-way ANOVA with Tukey’s correction. B, Female and male mice decreased time spent in the null zone after the first training day, one-way ANOVA with Tukey’s correction. All significances are in comparison with day 1. C, Female and male mice show an increase in ratio of time spent in the reward compared with null zone on day 2 of training, unpaired t test (two-tailed). Data are presented as the mean ± SEM; *p ≤ 0.05, **p ≤ 0.01.
Figure 4.
Figure 4.
Temperature-dependent tolerance is affected by sex and reward salience. A, Female (n =8) and male (n =8) mice differed in their tolerance to temperatures in the OPTA with 4% sucrose reward. Below, Representative heat maps of female and male mice in 30/45°C test. B, Male mice increased tolerance to aversive temperatures when 10% sucrose was available (n =7) compared with a reward of 4% sucrose (n =8). Below, Representative heat maps of 4% and 10% sucrose conditions. C, Female mice (n =8) exhibited reduced time in the reward zone when the reward was 10% sucrose compared with a reward of 4% sucrose across temperatures. Below, Representative heat maps of 4% and 10% sucrose conditions. D, Male mice (n =10 per group) spend more time in reward zone when 4% sucrose is present compared with when an empty bottle is present, unpaired t test (two-tailed). Furthermore, time spent in reward zone is significantly positively correlated with sucrose solution consumption. E, Pearson correlation, y = 185.4 + 114.8. F, Upon removal of the reward, male mice spend less time in the reward zone, paired t test (two-tailed). Note, male and female mice in 4% sucrose condition served as sex and age matched control for 10% sucrose condition. R = reward zone. A–C, Two-way ANOVA with Sidak’s correction. Data are presented as the mean ± SEM; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
Figure 5.
Figure 5.
Neuropathic pain increased time in reward zone and mobility in the OPTA. A, The radiant heat withdrawal assay showed decreased paw withdrawal latency in CCI versus sham (n =8 per group), unpaired t test (two-tailed). B, High and low temperatures were aversive for both CCI and sham animals, although CCI animals spent more time in the reward zone across even aversive temperatures, two-way ANOVA, Sidak’s correction. Below, Representative heat maps at 30/40°C test. C, Analysis of pooled time in the reward zone, paired t test, (two-tailed). D, CCI presented with greater distance traveled throughout testing, two-way ANOVA, Sidak’s correction. Below, Representative track plots at 30/15°C test. E, Analysis of pooled distance traveled, paired t test, (two-tailed). Data are presented as the mean ± SEM; **p ≤ 0.01, ****p ≤ 0.001. R = reward zone.
Figure 6.
Figure 6.
Neuropathic pain and clonidine in the OPTA. A, The radiant heat withdrawal assay showed clonidine increased withdraw threshold regardless of neuropathic pain, (n =12 per group), two-way ANOVA, Sidak’s correction. B, Clonidine increased time in reward zone in noxious heat chamber. Sham vehicle (n = 4); sham clonidine (n = 8); CCI vehicle (n = 10); CCI clonidine (n = 9), two-way ANOVA, Sidak’s correction. C, Representative heat maps of vehicle and clonidine treatment in sham and CCI. D, Neither clonidine nor CCI influence distance traveled, two-way ANOVA, Sidak’s correction. E, Representative tracking plots of vehicle and clonidine treatment in sham and CCI surgeries. Sham vehicle (n = 6); sham clonidine (n = 6); CCI vehicle (n = 8); CCI clonidine (n = 10). R = reward zone. Data are presented as the mean ± SEM; ****p ≤ 0.001.
Figure 7.
Figure 7.
Inflammatory pain in the OPTA. A, CFA (n =16) resulted in reduced withdrawal latency compared with sham (n =16) in the radiant heat withdrawal assay indicating the presence of hyperalgesia from CFA injection up to 48 h after injection, unpaired t tests (two-tailed). B, CFA did not affect time in reward zone in the temperature-dependent tolerance test, two-way ANOVA, Sidak’s correction. Below, Representative heat maps. C, CFA did not alter distance traveled at any specific temperature or across pooled temperatures, two-way ANOVA, Sidak’s correction. Below, Representative track plots. R = reward zone. Data are presented as the mean ± SEM; *p ≤ 0.05, **p ≤ 0.01.
Figure 8.
Figure 8.
Meloxicam in a model of inflammatory pain in the OPTA. A, There was a significant interaction between the effect of plantar injection and treatment on withdrawal latency in the radiant heat withdrawal assay, two-way ANOVA with Tukey’s correction. B, Both CFA and meloxicam reduced time in reward zone. C, Representative heat maps of vehicle and meloxicam treatment in control and CFA. D, CFA resulted in reduced distance traveled as compared with control, two-way ANOVA with Tukey’s correction. E, Representative track plots of vehicle and meloxicam in control and CFA. For all groups, n = 8. R = reward zone. Data are presented as the mean ± SEM; *p ≤ 0.05, **p ≤ 0.01.

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