Effects of hot and cold stimulus combinations on the thermal preference of rats

Abstract

Traditional evaluation of pain in animals has primarily used reflexive withdrawal or nocifensive response from singly presented stimulation. However, daily experience of thermal sensation involves situations in which rapid temperature changes from cold to hot can occur. Therefore, in order to better understand integration of competing stimuli and their role in the motivational character of pain perception, behavioral tasks have been adapted to evaluate treatment-driven changes in hindpaws when exposed to two or more stimuli. However, such assessments of craniofacial sensitivity are lacking. In this study, we sought to characterize thermal preference for facial stimulation when rats are given the option of experiencing a hot or cold stimulus to obtain a milk reward, or abstaining from stimulation. We found that when both cold and hot stimuli were either non-noxious or where both stimuli were noxious the hot stimulus was preferred. When the hot stimulus was noxious, non-noxious cold was preferred. Unstimulated time was dependent on the combined aversiveness of the two stimuli, such that unstimulated time was the greatest with a highly aversive stimulus pair (-4 and 48 degrees C). We also found that pairing stimuli modulated successful task completion for each stimulus, but for nociceptive heat, this was not solely a consequence of thermal preference. Finally, we found that previous preference could both induce and abolish subsequent thermode preference independent of stimulus cues. The findings in this study will allow us to evaluate experimental pain states and analgesic treatments in a manner more relatable to the experience of the patient.

Fig. 1. Distribution of time spent on the cold, hot, or off the thermode and percentage of licks spent at either thermode when cold stimuli were paired with 45 or 48°C

(A) More time is spent on the 45°C stimulus when paired with a 10, 18, or 24°C cold stimulus (see Table 2 for F statistics). Significantly less time is spent unstimulated (“Off”) when the cold stimulus is 24°C relative to the unstimulated time for the other two pairs (F _{2, 30} = 7.016). (B) The majority of licks also occur in contact with the 45°C stimulus (see Table 2), but there is no significant difference among licks in contact with cold thermode across stimulus pairs (F _{2, 30} = 1.192). (C) More time is spent on the non-noxious cold stimulus (10, 18, 24°C) when paired with 48°C. In contrast, more time is spent at 48°C when the cold stimulus is noxious (−4°C). The lowest percentage of unstimulated time occurs when the cold stimulus is 24°C and the greatest when it is −4°C (F _{3, 76} = 20.481, p<0.001). The percentage of time spent on the cold stimulus when it is −4°C is significantly less than the percentage of time on all other cold stimuli when paired with 48°C (F _{3, 76} = 20.481, p<0.001). (D) More licks occur at 10 or 18°C than at 48°C (although with 24°C as the cold stimulus this does not reach statistical significance), and the opposite is true when −4 and 48°C are paired (see Table 2). * indicates significant difference (i.e. preference) in time spent on either stimulus or unstimulated (repeated-measures ANOVAs with post-hoc LSD test) or between licks spent on the two stimuli (paired t-tests). + indicates a significant difference in percentage of licks or testing time across stimulus pairs for cold stimuli, hot stimuli, or unstimulated duration (one-way ANOVA with post-hoc Tukey's test). All data are mean ± SEM, significance is p < 0.05.

Fig. 2. Successful task completion when stimuli are paired or presented alone

(A) Success at 45°C is not significantly increased by pairing with non-noxious cold stimuli as compared to when it is presented alone (n = 29; F _{3, 62} = 2.457, p = 0.103). (B) Success ratios at the cold stimuli (10, 18, 24°C) are significantly decreased when paired with 45°C (10°C: t₅₁ = 12.264, for single stimulus, n = 41; 18°C: t₁₇ = 6.633, n = 12; 24°C: t₂₂ = 3.866, n = 14). (C) Success ratio at 48°C is significantly reduced when paired with −4 and 10°C, but not significantly different when paired with 18 or 24°C as compared to when it is presented alone (n = 28). (D) Success ratio is significantly decreased at −4 and 10°C when paired with 48°C (−4°C t₁₃ = 2.708; 10°C: t₁₀₅ = 2.706), significantly increased at 18°C (t₁₁ = −2.486), and not significantly increased at 24°C (t₉ = −1.46). * indicates p<0.05 for success alone versus paired, (for success ratios with hot stimuli significance was determined by ANOVA and Tukey's test post-hoc, and by t tests for success ratios with cold stimuli). All data are mean ± SEM.

Fig. 3. Preference can be conditioned or abolished by stimulus exposure 24 hours prior

(A) Two equivalent stimuli normally do not produce a preference, as indicated by 37 and 37°C (“NS” indicates no significant difference between thermodes). However, when preceded by a preference inducing combination (42 and 10°C; F_{2, 9} = 50.39), the next day more time is spent on the left 10°C thermode (previously preferred) than the right 10°C thermode (previously avoided, F_{2, 9} = 48.09). When comparing the right thermode across the stimulus pairs, the percentage of time spent on the right 10°C thermode was also significantly different from the right thermode set at 37°C (F_{2, 9} = 6.96). (B) Previous experience can also hinder the presentation of a stimulus driven preference. In this case, an avoidance of the left thermode (52°C) and preference for the right thermode (18°C) was established (F_{2, 8} = 91.28). The following day, the cold and hot sides were switched such that the left thermode was 18°C and the right, 48°C. Rats spend an equal percentage of time on the left and right thermodes (p = 0.909 for post-hoc LSD comparison of stimulus duration). In contrast, when tested two days later at with the same stimulus configuration, they exhibited a preference for the left 18°C stimulus (F_{2, 8} = 37.86). When comparing duration on the 18°C stimulus across these stimulus pairs, the time spent on 18°C (black bar) when paired with 52°C was significantly greater than the time spent on 18°C (grey bar) for the first 18/48°C pair (no preference), but not for the second 18/48°C pair (preference returned; F_{2, 8} = 10.73). * indicates within pair significant difference, p < 0.05, with repeated measures ANOVA and post-hoc LSD test. All data are mean ± SEM.