We developed a UV-exposure mouse model in which dorsally shaved mice received a dose of 50 mJ/cmof UVB 5 days per week for 6 weeks, an empirically derived suberythemic dose that is approximately equal to 20–30 min of ambient midday sun exposure in Florida during the summer for a fair-skinned person of average tanning ability (Fitzpatrick skin phototypes 2–3) (). After 1 week, significant elevations in circulating plasma β-endorphin were observed ( Figure 1 A). Circulating β-endorphin levels remained elevated for the duration of the 6-week exposure regimen and returned within 7 days to near-baseline levels after cessation of UV exposure. No significant changes in plasma β-endorphin were observed in mock-UV-treated mice ( Figure 1 A). Analgesic thresholds can be increased by peripheral administration of exogenous opioids or β-endorphin (). We quantified mechanical and thermal nociceptive thresholds over 6 weeks of daily UV exposure. Mechanical nociception was measured by the von Frey test (), which exposes fibers of increasing tensile strength to the plantar paw surface to elicit a paw-withdrawal response. Thermal nociception was tested using the hot-plate (52°C) test (), in which time to response (paw licking, paw flutter, or jumping) was measured. UV-irradiated mice exhibited significant increases both in mechanical ( Figure 1 B) and thermal ( Figure 1 C) nociceptive thresholds. These elevated analgesic thresholds paralleled the UV-induced elevations in plasma β-endorphin ( Figure 1 A). Mock-treated control mice displayed no significant elevations in pain thresholds ( Figures 1 B and 1C). Treatment with naloxone, an opioid antagonist, 15 min prior to analgesic testing suppressed the UV-induced increases in mechanical and thermal nociceptive thresholds ( Figures 1 B and 1C) despite maintained elevations in plasma β-endorphin ( Figure 1 A). These data demonstrate opioid receptor-mediated analgesia as a consequence of UV that parallels the elevation of circulating blood β-endorphin.

(B and C) Von Frey thresholds (B) and hot-plate thresholds (C) in chronically UV-irradiated and mock-irradiated C57Bl6 mice (mean ± SEM). Half of each group was pretreated with naloxone (10 mg/kg) 15 min prior to nociceptive testing, while the remainder received saline (n = 10 per group). Analgesic thresholds were further monitored for 2 additional weeks after cessation of UV/mock treatment. Two-way ANOVA with Bonferroni multiple comparisons test reveals p < 0.0001 for the UV/saline-treated group compared to all other groups during UV treatment, days 9–39).

(A) Plasma β-endorphin in C57Bl6 mice receiving daily UV or mock irradiation (n > 9 for all groups). Mice were treated twice a week with either naloxone or saline as indicated. Data are presented as the mean ± SEM, and a two-way ANOVA analysis with Bonferroni multiple comparisons test gives p < 0.05 for both UV-treated groups compared to both mock-treated groups (during UV treatment, days 14–42) and no significant effect of naloxone treatment within either group.

Exogenous opioids produce a dose-dependent, μ-opioid receptor-mediated contraction of the sacrococcygeus dorsalis muscle at the tail base in rodents, resulting in rigidity and elevation of the tail, a phenomenon called “Straub tail” (). Straub tail was evident in UV-irradiated mice by the second week of daily UV exposure, persisted for the 6 week exposure regimen, and diminished over 2 weeks after cessation of UV ( Figure 2 A). Treatment with the opioid antagonist naloxone (day 23 of the UV-exposure regimen) reversed the Straub tail phenotype ( Figures 2 B and 2C).

(B) Straub tail in at day 17 before (Pre) and 15 min after (Post) injection of naloxone (n = 7) or saline (n = 6). Data are presented as the mean ± SEM; p < 0.001 by Student’s t test.

(A) Straub tail in C57Bl6 mice over the course of 42 days of UV irradiation (n = 13) or mock irradiation (n = 6). Data are presented as the mean ± SEM for days 10–37; p < 0.0001 by two-way ANOVA analysis.

Opioid Tolerance and Physical Dependence after Chronic UV Exposure

Drdla et al., 2009 Drdla R.

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Palmiter R.D. Lack of neuropeptide Y attenuates the somatic signs of opiate withdrawal. Figure 3 Chronically UV-Exposed Mice Show Symptoms of Opioid Dependence Show full caption (A) Signs of opioid withdrawal in mice under experimental conditions described in the figure: UV/saline (n = 9), mock/saline (n = 7), UV/naloxone (n = 15), and mock/naloxone (n = 7). Data are presented as the mean ± SEM; ∗p < 0.05 compared to UV/saline group by two-way ANOVA with Bonferroni multiple comparisons test. (B) Conditioned place aversion testing in UV-treated mice conditioned to the naloxone-paired box (black box) with an injection of naloxone or saline (white box) following 42 days of UV or mock treatment. Mice were permitted to freely move between naloxone-paired and saline-paired boxes prior to (pretest, n = 8) and after 4 days of conditioning (test), and place preferences were assessed as change in time spent in the naloxone-paired box (postconditioning − preconditioning). Data are presented as the mean ± SEM, and p values were generated by two-way ANOVA with Bonferroni multiple comparisons test. (C) Morphine dose-response curves in mice following 42 days of UV irradiation (n = 31) or mock exposure (n = 29). Data are presented as the mean ± SEM; p < 0.0001 by two-way ANOVA. (D) Conditioned place-preference testing in mice administered intravenous β-endorphin or saline through the tail vein. Mice were conditioned to β-endorphin (n = 6) or saline (n = 8) in the white box and saline in the black box. Place preferences were assessed as change in time spent in the white (β-endorphin)-paired box, postconditioning − preconditioning. Data are presented as the mean ± SEM; p = 0.0145 by Student’s t test. We next asked whether chronic UV exposure may be accompanied by detectable opioid dependence, in which opioid cessation or antagonism produces withdrawal symptoms, and tolerance in which increasing doses are required to achieve comparable analgesia (). Following chronic daily UV exposure, administration of naloxone elicited many of the classic murine signs of opioid withdrawal (wet dog shake, paw tremor, teeth chatter, and rearing) () ( Figure 3 A).

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Inturrisi C.E. Conditional deletion of the NMDA-NR1 receptor subunit gene in the central nucleus of the amygdala inhibits naloxone-induced conditioned place aversion in morphine-dependent mice. Kenny et al., 2006 Kenny P.J.

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Riley A.L. Apparatus bias and the use of light and texture in place conditioning. Because the magnitude of the measured withdrawal symptoms, while significant, was smaller than that commonly observed with exogenously administered opioids (), we wished to determine whether these withdrawal signs would be sufficient to elicit alterations in proactive/operant behavioral choices. We utilized a conditioned place preference/aversion assay (CPP;) to test whether a specific environment, paired with naloxone administration during conditioning, would be avoided in favor of a different environment paired with a neutral stimulus (saline) during conditioning in chronically UV-irradiated animals. Due to the kinetics of the UV response, we chose to use naloxone as it allowed an acute effect of limited duration. Naloxone induces conditioned place aversion in exogenous opioid-dependent mice (). Following conditioning, mice are permitted to move freely between the two environments, and changes in place preference are measured in the absence of additional naloxone or saline administration. Our conditioning environments were black and white boxes with dim and bright lighting, respectively, and to minimize apparatus bias, we assigned the black box as the naloxone (withdrawal stimulus)-paired box and the white box as the saline (neutral stimulus)-paired box, as rodents prefer dark environments to light environments in the absence of conditioning ().

We observed that chronically UV-irradiated mice conditioned with naloxone in the black box avoided the black box in postconditioning preference testing. Naloxone conditioning had no effect on mock-treated (non-UV-irradiated) control mice, and saline conditioning in the black box had no effect on UV-irradiated or mock-treated mice ( Figure 3 B). Here, naloxone was sufficient to induce conditioned place aversion in UV-irradiated mice, suggesting that chronic UV exposure imparts an opioid-like physical dependence of sufficient magnitude to guide proactive behavior choices.

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Li C.H. Beta-Endorphin as a potent analgesic by intravenous injection. To test for the other principal feature of chronic opioid exposure, tolerance, after chronic UV treatment, we asked whether there is cross-tolerance between chronic UV exposure and morphine, altering the dose required to produce analgesia (). After chronic UV exposure, mice required significantly higher doses of morphine than mock-treated controls to achieve comparable thermal analgesia in the hot-plate test, as reflected by a rightward shift in the dose-response curve and an increase in half-maximal effective concentration from 57 μg/kg in the mock-treated group to 270 μg/kg in the UV-exposed group ( Figure 3 C). The analgesic effect of UV exposure that we detected could be a result of systemic β-endorphin acting both through the peripheral nervous system and CNS, but the withdrawal effects and conditioned place aversion point to a CNS effect. It has been reported that radiolabeled β-endorphin peptides cross the blood-brain barrier (). To test whether it is plausible that skin-derived β-endorphin may cause central effects, we decided to assess whether peripherally administered β-endorphin injected intravenously into the tail vein could cause conditioned place preference. To attempt to match an acute intravenously administered drug dose with a chronic elevation, we chose a βendorphin concentration reported to cause a similar analgesic response to that we observed in our UV-exposure experiments, (). β-endorphin or saline was injected into the tail vein of mice that were then conditioned to the white side of the CPP apparatus. The mice that had been conditioned with saline spent less time in the white box on the final day than on the initial day ( Figure 3 D); this was expected, as mice naturally prefer a dark environment. However, the mice that had received β-endorphin in the white box spent more time in the white box after conditioning ( Figure 3 D), indicating a conditioned place preference for the environment where they experienced β-endorphin. This shows that peripherally administered β-endorphin can cause conditioned place preference, presumably through the CNS.

These findings show that chronic UV exposure stimulates and sustains sufficient endogenous opioid release and opioid receptor activity to develop both opioid tolerance and physical dependence.