Contextual control of conditioned pain tolerance and endogenous analgesic systems
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Evaluation Summary:
This study covers a series of experiments designed to characterize conditioned pain processing using a novel animal model in which mechanical nociception (von Frey test) is assessed following exposure to contextual cues that have been paired with visceral pain (intraperitoneal acid injection). These experiments address an important topic from a translational perspective, both because learning is an important but understudied contributor to the human pain experience and because there is evidence for sex differences in human pain expression. The results reveal that such cues exert complex, dose- and sex-dependent effects on pain processing that will be of broad interest to researchers across fields of associative learning, neuroscience, and pain research.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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Abstract
The mechanisms underlying the transition from acute to chronic pain are unclear but may involve the persistence or strengthening of pain memories acquired in part through associative learning. Contextual cues, which comprise the environment in which events occur, were recently described as a critical regulator of pain memory; both male rodents and humans exhibit increased pain sensitivity in environments recently associated with a single painful experience. It is unknown, however, how repeated exposure to an acute painful unconditioned stimulus in a distinct context modifies pain sensitivity or the expectation of pain in that environment. To answer this question, we conditioned mice to associate distinct contexts with either repeated administration of a mild visceral pain stimulus (intraperitoneal injection of acetic acid) or vehicle injection over the course of 3 days. On the final day of experiments, animals received either an acid injection or vehicle injection prior to being placed into both contexts. In this way, contextual control of pain sensitivity and pain expectation could be tested respectively. When re-exposed to the noxious stimulus in a familiar environment, both male and female mice exhibited context-dependent conditioned analgesia, a phenomenon mediated by endogenous opioid signaling. However, when expecting the presentation of a painful stimulus in a given context, males exhibited conditioned hypersensitivity whereas females exhibited endogenous opioid-mediated conditioned analgesia. These results are evidence that pain perception and engagement of endogenous opioid systems can be modified through their psychological association with environmental cues. Successful determination of the brain circuits involved in this sexually dimorphic anticipatory response may allow for the manipulation of pain memories, which may contribute to the development of chronic pain states.
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Evaluation Summary:
This study covers a series of experiments designed to characterize conditioned pain processing using a novel animal model in which mechanical nociception (von Frey test) is assessed following exposure to contextual cues that have been paired with visceral pain (intraperitoneal acid injection). These experiments address an important topic from a translational perspective, both because learning is an important but understudied contributor to the human pain experience and because there is evidence for sex differences in human pain expression. The results reveal that such cues exert complex, dose- and sex-dependent effects on pain processing that will be of broad interest to researchers across fields of associative learning, neuroscience, and pain research.
(This preprint has been reviewed by eLife. We include the public …
Evaluation Summary:
This study covers a series of experiments designed to characterize conditioned pain processing using a novel animal model in which mechanical nociception (von Frey test) is assessed following exposure to contextual cues that have been paired with visceral pain (intraperitoneal acid injection). These experiments address an important topic from a translational perspective, both because learning is an important but understudied contributor to the human pain experience and because there is evidence for sex differences in human pain expression. The results reveal that such cues exert complex, dose- and sex-dependent effects on pain processing that will be of broad interest to researchers across fields of associative learning, neuroscience, and pain research.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
Trask et al. first investigated whether males and females develop a conditioned compensatory response to pain-associated contexts: an effect that has been shown previously to discrete stimuli, and predominantly in male mice. In their first two experiments, they found that both males and females can indeed develop such a response, but that females do so more readily, with lower dose acetic acid injections used in the training phases. They then show that these effects are likely mediated by endogenous opioids rather than corticosterone, because they are blocked by systemic injections of the opioid-antagonist naloxone but have no relation to levels of corticosterone. Finally, they attempt to tease apart whether these effects are truly 'conditioned' or a result of some autonomic response to the acetic acid …
Reviewer #1 (Public Review):
Trask et al. first investigated whether males and females develop a conditioned compensatory response to pain-associated contexts: an effect that has been shown previously to discrete stimuli, and predominantly in male mice. In their first two experiments, they found that both males and females can indeed develop such a response, but that females do so more readily, with lower dose acetic acid injections used in the training phases. They then show that these effects are likely mediated by endogenous opioids rather than corticosterone, because they are blocked by systemic injections of the opioid-antagonist naloxone but have no relation to levels of corticosterone. Finally, they attempt to tease apart whether these effects are truly 'conditioned' or a result of some autonomic response to the acetic acid injection (by which I assume they mean some kind of habituation process) by omitting the acetic acid injection prior to test. The results here are somewhat confusing, but appear to demonstrate that in females at least, the effect is truly conditioned - i.e. the compensatory response is present even when the animals do not receive acid injections prior to test.
Overall this is a well-written and well presented manuscript that reports a series of interesting and novel results in an elegant manner. For the most part, the conclusions appear well supported, although I do have some queries as listed in the weaknesses section below. The results are particularly illuminating with regards to how important it is to include both female and male mice in our experiments - a practice that is still not ubiquitous - and illustrate that any conclusions made on the basis of experiments with males only may not generalise well to females. I think the results of this paper will be of interest to many researchers across fields of associative learning, neuroscience, and pain research.
Strengths:
• The manuscript has many strengths. It addresses an important novel topic, presents some exciting results, and presents the results in a systematic way.
• It is well written, the sample sizes and statistics are appropriate for the most part, and the discussion is insightful.
• The conclusions are mostly well supported although I do have some questions regarding whether they are all supported, which I will list in the weaknesses section.Weaknesses
1. For the most part the logical flow of the manuscript is good, however, there are some sections in which I found the organisation difficult to follow. For example, Figure 1 details two experiments: one in which context-pain associations are formed and tested, and another in which animals are given naloxone. In the text, the first two experiments described involve escalating or consistent dose acetic acid injections, no mention of naloxone. The design is in Figures 2 and 3. Do the authors mean that Figure 1 explains two experimental series, each of which include multiple experiments? In addition, the manuscript jumps from explaining clear sex differences in conditional pain tolerance to reporting naloxone experiments for which results appear to be averaged across sexes, which is not signposted and thus a bit jarring.
2. Figure 3D. The authors suggest that, because male mice had higher withdrawal thresholds in the acid-paired context than in the vehicle context on day 4, that these animals were able to develop context-dependent pain tolerance. However, the fact that there is no difference in responding in this Context on Days 1 and 4 counteracts this claim somewhat - if animals learned tolerance across days then thresholds should be higher on Day 4. I also note that there is a lot of variability in the data, particularly with some animals increasing and some decreasing. I wonder if this works similarly to punishment - whether there is a median split of animals who develop tolerance and those who don't. It is quite the contrast from the females who are all in the same direction over days.
3. The corticosterone data (Lines 303-306, Figure 4C) is correlational but is referred to as causal (e.g. "...tolerance is not mediated by changes in circulating corticosterone...").
4. The null effect in the vehicle group in Figure 4X relies on a very low sample size, n = 4.
5. I found the experiments reported in Figure 5 a little confusing. Males appear to maintain their sensitivity to the context, suggesting the lack of a development of a compensatory mechanism in these mice, which is contradictory to the authors' claims in prior sections. Moreover, I'm not clear on why the data in Figure 5E show that females exhibit conditional recruitment of endogenous opioid systems in the training context? For this to be true, it would seem to me that the authors need to show that this effect (i.e. the naloxone-mediated increase in sensitivity) is not present in the vehicle context also.
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Reviewer #2 (Public Review):
As noted by the authors, there has been relatively little research on the basic mechanisms of how context cues influence pain processing, particularly outside the fear learning literature. The current study addresses this gap in knowledge by developing a robust and reliable animal model for this phenomenon.
The experimental design is generally appropriate for the question at hand and has a number of strengths that should make it attractive to researchers in this area. Most notably, it has a short experimental timeline (3 days of conditioning and 1 day of testing) and makes use of well established procedures for inducing visceral pain (acetic acid injection) and assessing sensitivity to mechanical irritation (von Frey filament withdrawal). While these individual preclinical pain models have been extensively …
Reviewer #2 (Public Review):
As noted by the authors, there has been relatively little research on the basic mechanisms of how context cues influence pain processing, particularly outside the fear learning literature. The current study addresses this gap in knowledge by developing a robust and reliable animal model for this phenomenon.
The experimental design is generally appropriate for the question at hand and has a number of strengths that should make it attractive to researchers in this area. Most notably, it has a short experimental timeline (3 days of conditioning and 1 day of testing) and makes use of well established procedures for inducing visceral pain (acetic acid injection) and assessing sensitivity to mechanical irritation (von Frey filament withdrawal). While these individual preclinical pain models have been extensively characterized in mice, the current study investigates how they interact, specifically how contextual cues paired with visceral pain influence the withdrawal reflex to an anatomically distinct touch stimulus.
Importantly, for most of the experiments in the manuscript (but see below), testing was conducted 45-60 min after an acetic acid injection, which was either coupled with exposure to a context that had already been paired with such injections or with a neutral (vehicle-paired) context. Findings from these experiments therefore reflect the unconditioned effects of visceral pain on mechanical touch sensitivity (apparent on the first day of conditioning following exposure to initial context-acid pairing or on the test day after exposure to the vehicle-paired context) and how these effects are modulated by context conditioning (apparent on the test day after exposure to the acid-paired context). The first experiment found that mice display mechanical allodynia (heightened sensitivity to touch stimuli) after visceral pain in the absence of pain-paired cues. Importantly, this pain-induced allodynia was abolished by a conditioned analgesic response (restoring withdrawal thresholds to baseline values) in females but not males when pre-test pain was signaled by pain-paired contextual cues. Follow-up experiments demonstrated that male mice show a similar albeit less pronounced conditioned analgesic effect at test when a more painful stimulus is used during conditioning, indicating that a sex difference in pain sensitivity may underpin the sex difference in conditioned analgesia described above.
While a within-subjects design was used in most experiments, the authors replicated their basic findings using a between-subjects design, which helps rule out more complex interpretations of the data and establishes the robustness and reliability of the basic findings. The authors also show that, after the test session, circulating levels of the stress-hormone corticosterone were similar for vehicle- and acid-trained mice, suggesting that the conditioned analgesic response described above was not mediated by this response, though it must be noted that there was no investigation of the time course of this effect and that context-dependent effects on corticosterone may have been obscured by nonspecific effects of pre-test acid injection and von Frey testing.
However, the conditioned analgesia induced by the pain-paired context was largely abolished when mice were pretreated with naloxone, indicating that this effect was mediated by an endogenous opioid response. This disruption was observed in both males and females. It was also observed in the final experiment, which contrasted the influence of pain- and vehicle-paired context cues alone, in the absence of visceral pain. Under these conditions, male mice actually showed conditioned allodynia (faster withdrawal) response when tested following exposure to the pain-paired context (relative to the neutral context). Females, on the other hand, showed no difference in withdrawal between contexts. However, this lack of cue-specificity was not due to a failure to learn or discriminate, but instead appears to reflect the engagement of two distinct conditioning processes, one that facilitates withdrawal (displayed by males) and a conditioned analgesic response which opposes this facilitation (not displayed by males). This was demonstrated by pre-treating mice with naloxone prior to testing, which selectively abolished the conditioned analgesic response in females, such that both sexes showed a conditioned allodynia effect.
It is important to note that the current study provides only the first step in establishing this new model, and therefore many important questions remain unanswered. For instance, von Frey testing was conducted after exposure to the pain-paired context. Thus it remains unclear whether a different result would be observed if the cues were actually present when sensitivity to nociceptive touch was actually being assessed. Similarly, it is not clear how long-lasting the conditioned effects persist after cue removal. These are important questions that have both practical and theoretical implications.
With regard to theory, opponent process accounts of pain processing have been advanced to explain conditioned analgesic effects like the one reported here, but do not readily explain the conditioned allodynia displayed by both sexes in the final experiment, when pre-test visceral pain was omitted. The current findings highlight the complexity of conditioned pain processing and raise important questions about the stimulus-specificity of such effects. For instance, it is unclear why male mice showed evidence of conditioned analgesia when given an injection of acid prior to testing but not when the paired-context was presented alone. Presumably it is that the conditioned analgesic effect is somewhat pain-specific, in that it is more effective in opposing the facilitatory influencing of pre-test visceral pain on nociceptive touch perception than it is in dampening nociceptive touch perception, per se.
These questions do not detract from the impressive work laid out here and instead represent goals for future research. There are, however, a few issues with the current study that make data interpretation difficult and weakens the overall impact of the paper in its present form. For instance, there is no description of writhing behavior (unconditioned pain response to acid injection) or how it may have interacted with von Frey testing. Moreover, the question of stimulus-specificity could be more directly addressed by assessing how the pain-paired context influenced writhing to the pre-test acid injection. Moreover, was there any evidence of conditioned effects of the pain-paired context on locomotor behavior and could this impact von Frey testing.
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Reviewer #3 (Public Review):
This study used a classical-conditioning design to compare effects of associative learning on expression of pain-related behaviors in male and female mice. The experiments address an important topic, both because learning is an important but understudied contributor to the human pain experience and because there is evidence for sex differences in human pain expression. In this study, IP injection of acetic acid served as the principal unconditioned stimulus (US), and hypersensitivity of hindpaw-withdrawal responses to mechanical stimulation was the unconditioned response (UR). The US or its vehicle control was paired with a test chamber as the conditioned stimulus (CS), and US+CS pairing produced changes in mechanical sensitivity that were interpreted as the conditioned response (CR).
There were three main …
Reviewer #3 (Public Review):
This study used a classical-conditioning design to compare effects of associative learning on expression of pain-related behaviors in male and female mice. The experiments address an important topic, both because learning is an important but understudied contributor to the human pain experience and because there is evidence for sex differences in human pain expression. In this study, IP injection of acetic acid served as the principal unconditioned stimulus (US), and hypersensitivity of hindpaw-withdrawal responses to mechanical stimulation was the unconditioned response (UR). The US or its vehicle control was paired with a test chamber as the conditioned stimulus (CS), and US+CS pairing produced changes in mechanical sensitivity that were interpreted as the conditioned response (CR).
There were three main findings. First, the IP-acid US was more potent to elicit mechanical-hypersensitivity as the UR in females than males. Second, pairing of the CS with US intensities adequate to elicit the UR resulted in a CR, so this study provides strong evidence of conditioning. In the presence of the US (i.e. in the presence of US+CS after US+CS pairing), the CR manifested as a reduction in mechanical-hypersensitivity UR in both sexes. This was interpreted as "conditioned pain tolerance." However, in the absence of the US (i.e. CS alone after US+CS pairing), the CR manifested as conditioned mechanical hypersensitivity only in males. Lastly, studies with naloxone implicated endogenous opioid signaling in conditioned pain tolerance in both sexes and resistance to conditioned mechanical hypersensitivity in females.
This study provides a novel approach to the use of classical conditioning for research on expression of pain-related behaviors. The intent was to identify patterns of learned pain behaviors in mice that both correlate with human patterns of pain expression and provide a basis for follow-up mechanistic studies. It is unclear if the results meet the translational goal, but the study describes novel strategies and outcomes of classical conditioning with noxious stimuli that are of high significance and warrant further study.
STRENGTHS
-The study used an appropriate experimental design to evaluate the degree to which contextual stimuli could be established as a CS to modulate expression of a pain-related behavior in male and female mice as the CR.
-Of particular importance, the study evaluated effects of conditioning on pain behavior not only in the absence of the pain stimulus (i.e. after presentation of the contextual CS alone) but also in the presence of the pain stimulus (i.e. after presentation of the pain stimulus US and contextual CS together).
-Results provide clear evidence of a sex difference in both acute effects of the pain stimulus on the pain behavior (i.e. the UR) and in conditioning produced by repeated presentation of that stimulus (i.e. the CR).
WEAKNESSES
-The pain stimulus used in this study (intraperitoneal acid injection) elicits a wide range of pain behaviors as URs. This study focused on one of these pain behavior URs (mechanical hypersensitivity of hindpaw withdrawal responses), and the sex difference in this UR (females more sensitive than males) appears to be substantially larger than for many other IP acid-induced URs (where sex differences are generally not significant). The basis for this larger sex difference is not considered, and the implications of this sex difference for conditioning of this behavior or for conditioning of other IP acid-induced behaviors are also not fully considered.
-Studies with the opioid antagonist naloxone implicate recruitment of endogenous opioid signaling as contributor to conditioned pain behaviors. However, an important control experiment with naloxone was omitted from one part of the study.
-The authors note that associative learning like that studied here may contribute to the transition from acute to chronic pain, and that chronic pain is more prevalent in women than men. However, conditioning effects observed in this study were protective in females. Consequently, it is not clear how results of this study might provide insight into the basis for higher prevalence of chronic pain in women.
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