- Email: [email protected]
Adolescent morphine induces emotional signs of withdrawal paired with neurotoxicity selectively in male rats: Female resilience
Journal Pre-proof Adolescent morphine induces emotional signs of withdrawal paired with neurotoxicity selectively in male rats: female resilience ´ Fernando Jimenez-Romero, Cristian Bis-Humbert, M. Julia Garc´ıa-Fuster
PII:
S0304-3940(19)30728-1
DOI:
https://doi.org/10.1016/j.neulet.2019.134625
Reference:
NSL 134625
To appear in:
Neuroscience Letters
Received Date:
3 September 2019
Revised Date:
8 November 2019
Accepted Date:
9 November 2019
´ Please cite this article as: Jimenez-Romero F, Bis-Humbert C, Garc´ıa-Fuster MJ, Adolescent morphine induces emotional signs of withdrawal paired with neurotoxicity selectively in male rats: female resilience, Neuroscience Letters (2019), doi: https://doi.org/10.1016/j.neulet.2019.134625
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
F. Jiménez-Romero, C. Bis-Humbert, M.J. García-Fuster
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Research Article NSL-191417-R1
Adolescent morphine induces emotional signs of withdrawal paired with neurotoxicity selectively in male rats: female resilience
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Fernando Jiménez-Romero, Cristian Bis-Humbert, M. Julia García-Fuster IUNICS, University of the Balearic Islands, and IdISBa, Palma, Spain
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Short title: Sex-specific effects of adolescent morphine in rats
Correspondence to: Dr. M. Julia García-Fuster. IUNICS, University of the Balearic Islands, Cra. de Valldemossa km 7.5, E-07122 Palma, Spain.
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Phone: +34 971 259992 Email: [email protected]
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Highlights
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Fax: +34 971 259501
Adolescent morphine induced persistent sex-dependent changes in negative affect.
Adolescent morphine induced persistent sex-dependent changes in brain toxicity.
Female rats seemed resilient to the negative effects induced by adolescent morphine.
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Abstract This study evaluated the immediate and persistent behavioral and molecular consequences (i.e., emotional signs emerging during withdrawal and signs of neurotoxicity) following adolescent
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morphine exposure with a sex perspective. Basally, and prior to drug treatment, adolescent female rats showed smaller body weight and lower pain threshold than their male counterparts. Adolescent morphine treatment induced some sex-specific differences, while morphine impaired normal weight gain in male rats, no effects were observed for female rats. Plus, morphine produced an attenuated antinociceptive response in female rats. Moreover, following adolescent morphine treatment some emotional signs of withdrawal emerged exclusively in male rats in conjunction with signs of neurotoxicity, while female rats were not affected. In
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particular, an anxiolytic-like effect in adolescence during early withdrawal was followed by the development of a depressive-like phenotype (i.e., behavioral despair, anxiety-like behavior and anhedonia) in adulthood during prolonged withdrawal and paired with decreased contents
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of neurofilaments proteins in the prefrontal cortex. In conclusion, morphine administration during adolescence induced persistent changes in negative affect and brain toxicity selectively
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in male rats, suggesting female rats were resilient to these harmful effects. Given the
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widespread availability and use of opiate-based painkillers, the interplay between addiction, analgesia and emotional behaviors, and since adolescents and young adult humans are the age group with the highest abuse potential, these results add to the current literature by reporting
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Keywords
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distinct sex-specific opioid actions when administered in adolescence.
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Sex differences; morphine; adolescence; withdrawal; age.
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1. Introduction Opioids accounted for 70% of the negative health impact associated with drug use disorders worldwide 1, being adolescents and young adult humans aged 12-25 years old more likely than any other age group to abuse illegal opioids or misuse prescription pain relievers 2. Studies in adolescent rodent models suggest that the response to opiates changes during development (e.g., 3). For example, adolescent rats self-administer less morphine than adults, an effect that was interpreted as higher drug sensitivity, and show lower rates of reinstatement,
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suggesting adolescent development might include some protective factors that diminish the long-term impact of early drug intake 4. This seems particularly relevant given that an early onset of opioids use has been associated with an increased chance of having a physical or
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psychiatric co-morbidity 5. In this context, opioid withdrawal in humans and rodents is accompanied by the precipitation of negative affect, such as anxiety, anhedonia, and aversion
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6-9. While these emotional signs of withdrawal tend to be experienced earlier and less
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severely in adolescent rodents than in their adult counterparts (e.g. 10), these studies have been mainly centered in the alcohol and nicotine fields. Therefore, age-related differences in
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affective responses to morphine withdrawal might explain the increased risk of mental illness when drug use begins during adolescence.
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Sex must be also considered to understand opioid use disorder (see commentary in 11). For instance, studies in humans and rodents suggest that females may be at greater risk for the
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nonmedical use of prescription opioids than for the use of heroin 12, while also appear to shift faster from a casual use of opioids to dependence 13,14, and show higher levels of craving and relapse during abstinence (see revision in 15), suggesting sex-specific factors for addiction liability related to differences in opioid signaling in the brain. Opiate withdrawal might affect mood differentially across sexes (e.g., 16,15). In this sense, sex differences in affective responses to withdrawal might contribute to the differential development of comorbid
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mental disorders in female and male rodents. However, there is a lack of studies on sex differences in affective-like responses to opioid withdrawal, especially during adolescence. Thus, the first aim of this study was to determine sex-based differences in the emotional signs of withdrawal that emerge following an escalating repeated morphine regimen in rats during adolescence. To date, the molecular mechanisms underlying morphine withdrawal have been mainly studied in male adult animals. In this context, prolonged opiate exposure is known to induce
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marked decreases in brain cytoeskeletal proteins, such as decreases in neurofilament (NF) proteins in the prefrontal cortex (PFC) of rats 17,18 and of opiate addicts 19, suggesting that such plastic changes may reflect the induction of cortical neuronal damage 20. However,
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when looking more into the specifics of these studies and of the prior literature, the reports have been centered in males, for example in 19, 16 out of 17 human subjects were males and
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only 1 female was included in the study. Moreover, and to be the best of our knowledge, no
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prior experiments evaluated the effects of morphine on NF protein content in female rodents or following adolescent morphine exposure. In this context, the second aim of this study was
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to determine sex-specific neurotoxicity induced by adolescent exposure that could accompany the sex-specific changes in emotional behavior during drug withdrawal. A preliminary report of this work was presented at the 31st European College of Neuropsychopharmacology
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Congress 21.
2. Material and methods 2.1. Animals
A total of 36 Sprague-Dawley rats (22 male and 14 female) from 3 litters bred at the same time in the animal facility at the University of the Balearic Islands were used. Rats were housed in standard cages (groups of 2 to 3) under precise environmental conditions such as 22 °C, 70%
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humidity, and 12 h light/dark cycle (lights on at 8:00 AM) and with ad libitum access to a standard diet and tap water. Animal experiments complied with ARRIVE guidelines 22 and EC Directive 86/609/EEC. All experimental procedures were performed during the light period and were previously approved by the Spanish Royal Decree 53/2013, the Local Bioethical Committee (University of the Balearic Islands) and the regional Government (Conselleria Medi Ambient, Agricultura i Pesca, Direcció General Agricultura i Ramaderia, Govern de les Illes
2.2. Behavioral testing and pharmacological treatment
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Balears).
Emotional state measures (i.e., core symptoms of a depressive-like phenotype) were evaluated
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by 3 consecutive experimental tests, forced-swim test (FST), novelty suppressed feeding (NSF) and sucrose preference (SP) that measure changes in behavioral despair, anxiety-like behavior
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and hedonic-like responses respectively. After weaning and prior to any drug treatment, some
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basal measurements were taken (see details in Fig. 1a). Rats were weighted on postnatal days (PND) 26, 29, 31 and 35. They were exposed to the stress of the FST, which consisted of a 15 min pre-test on PND 27 followed by a 5 min test on PND 28 (this session was videotaped).
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Briefly, each rat was individually placed in a tank (41 cm high x 32 cm diameter), with water to a depth of 25 cm, and at 25 1 C as described before in 23. Videos were blinded for
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analysis to score individual performances in the basal time spent immobile (sec) (Behavioral
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Tracker software, CA, USA). Then, rats were exposed to the NSF (see 24). To do so, rats were food deprived for 48 h (starting on PND 29) prior to testing on PND 31 in an open-field apparatus (square arena: 60 x 60 cm and 40 cm of height) with a small piece of rat chow placed in the center of the arena. Each animal started in a corner of the arena and was allowed a maximum of 5 min to begin feeding. Sessions were recorded and videos were analyzed blind to the experimental groups using a digital video tracking system (Smart Video Tracking
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software, Version 3.0.03, Panlab SL, Barcelona, Spain) to calculate the latency to feed (sec) as well as the feeding time (sec). After testing, animals were placed in their home cage with food and water ad libitum. Finally, the tail-flick test (TF) was performed on PND 37 as described earlier 18 to measure basal latencies for each rat (i.e. average of 3 individual measurements for each rat) one day prior to treatment. The TF latency was defined as the time from the onset of radiant heat to tail withdrawal. To minimize tail skin tissue damage, cutoff time was set as 3 times the baseline latency.
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Then, rats were repeatedly treated during mid-adolescence (3 times/day, for 4 days, from PND 38-41 and 2 times on PND 42, spaced 5-6 hours during the lights on period, i.p.; see revision in 25 for developmental stages for rodents) with saline (0.9% NaCl, 1 ml/kg, n=11
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for the male group and n=7 for the female group) or with an escalating regimen (10-100 mg/kg, n=11 for the male group and n=7 for the female group) of morphine HCl (Unión Químico-
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Farmacéutica S.A.E., Madrid, Spain). The daily doses of morphine were based on prior studies
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from our research group (e.g., 26): day 1, 10/10/10 mg/kg; day 2, 10/20/20 mg/kg; day 3, 20/20/40 mg/kg; day 4, 40/40/80 mg/kg; and day 5, 80/100 mg/kg. The TF was used to evaluate
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the antinociceptive response of morphine on PND 38, 40 and 42 (days 1, 3 and 5 of treatment respectively) as measured 1 h after the first daily injection. Consecutive behavioral tests were
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then performed to evaluate negative affect during immediate (PND 45-64) and prolonged withdrawal (PND 70-84, following an analgesic challenge dose of 10 mg/kg of morphine
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administered to all 36 rats to compare the acute effects of morphine in naïve rats vs. its effects in the FST in rats previously exposed to morphine in adolescence on PND 70): (1) FST to evaluate behavioral despair (a 5 min test on PND 45 and PND 70); (2) NSF for anxiety-like behavior (a 5 min test on PND 47 and PND 72 following food removal 48 h prior to test); and (3) 1% SP (two bottle choice) test for hedonic-like response (a 48 h test on PND 62-63 and PND 82-83) (see further details in 27,28). Rats were single housed starting on PND 56 (to
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evaluate the individual hedonic response) and remained like that until the end of the experiment. This experimental design was set to evaluate the temporal course of the behavioral effects emerging during morphine withdrawal. To avoid the effects of learning and/or the development of tolerance to the test performance due to repetition of the behavioral tests, the same conditions were followed for all rats, in an attempt to compare the progression of treated rats vs. their respective controls (see 27 for similar procedures).
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2.3. Tissue collection for western blot analysis
Rats were sacrificed on PND 89 (Fig. 1a), the PFC was freshly dissected, immediately frozen in liquid nitrogen and stored at -80 C until tissue was prepared for western blot analysis
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following standardized lab protocols: 40 μg of total protein for each sample were resolved by electrophoresis, transferred to a nitrocellulose membrane and incubated with selected
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antibodies (e.g., 29). Information about primary antibodies, previously characterized by our
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group 18,19,29, regarding vendors and dilution conditions: (1) Covance (CA, USA): antiNF-H and M (clone SMI-32; 1:1000); and (2) Sigma-Aldrich (MO, USA): anti-NF-L (N5139;
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1:1000), anti-ß-actin (clone AC-15; 1:10000). The corresponding secondary antibodies (antirabbit or anti-mouse IgG linked to horseradish peroxidase) were incubated for 1 h at room
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temperature (1:5000 dilution; Cell Signaling). Immunoreactivity of target proteins was detected with ECL reagents (Amersham, Buckinghamshire, UK) and signal of bound antibody
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was visualized by exposure to autoradiographic film (Amersham ECL Hyperfilm) for 1 to 60 min, which was quantified by densitometric scanning (GS-800 Imaging Calibrated Densitometer, Bio-Rad). Percent changes in immunoreactivity were calculated for each rat in each gel with respect to male control samples (M-Sal: 100%). Each rat sample was run at least 2-3 times in different gels, and the mean value was used as a final estimate. The content of ßactin was used as loading control.
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2.4. Statistical analyses Data were analyzed with GraphPad Prism, Version 8 (GraphPad Software, Inc.). Results are expressed as mean values ± standard error of the mean (SEM). Two (Sex and Day) or threeway (Treatment, Sex and Day) ANOVAs (with or without repeated measures, RM) followed by Sidak's multiple comparisons test when appropriate or two-tail Student's t-tests (for changes
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in NF content) were used as statistical analyses. The level of significance was set at p≤0.05.
3. Results 3.1. Basal measurements
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As expected, there were basal body weight differences in male and female offspring (Sex x Day interaction: F3,102=10.89, p<0.001; Fig. 1b). In particular, female rats weighted less than
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their male siblings at any time point evaluated (*p<0.05; **p<0.01 and ***p<0.001). When
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evaluating behavioral despair (time spent immobile) in the FST (PND 28), no basal differences between male and female rats were observed (t=1.46, df=34, p=0.152; Fig. 1b). Similarly, no basal changes were observed for anxiety-like behavior (latency to feed in the NSF; t=0.41,
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df=34, p=0.684; Fig. 1b) on PND 31. However, when basal pain response was evaluated in the TF on PND 37, female rats showed a reduced latency to remove the tail from the hot laser
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exposure (-0.970.33 sec vs. male rats; t=2.89, df=34, **p<0.01; Fig. 1b).
3.2. Effects of treatment on body weight As shown in Fig. 1c, when measuring the effects of treatment on body weight there was a significant effect of Treatment (F1,32=8.91, p<0.01), Sex (F1,32=215.4, p<0.001), Day
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(F1.742,55.73=2863, p<0.001) and Sex x Day interaction (F8,256=293.9, p<0.001). In particular, when evaluating the effect of Treatment male rats treated with morphine showed reduced normal weight gain following 4 days of treatment (PND 41; -237.5 g, *p<0.05 vs. salinetreated rats), an effect that remained significant throughout the rest of the study (from PND 4780, range of smaller body weight from -20 to -247.5 g, *p<0.05, Fig. 1c). Female rats continued to be smaller than their male counterparts and showed no changes by Treatment.
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3.3. Effects of treatment on antinociception
During drug treatment (PND 28-42), the antinociceptive effects of morphine were evaluated in the TF test on PND 38, 40 and 42 (days 1, 3 and 5 of treatment respectively). There was a
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significant effect of Treatment (F1,32=166.4, p<0.001) and Sex (F1,32=3.99, p=0.05), but not of Day (F2,64=0.92, p=0.404). In particular, although the effect of Treatment was observed for
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male and female rats (i.e., increased TF latency by morphine vs. saline-treated rats;
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***p<0.001; Fig. 1d) at all days measured, female rats showed a smaller increase induced by
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morphine (e.g., -1.55 0.49 sec on PND 42 vs. male morphine-treated rats, #p < 0.01, Fig. 1d).
3.4. Effects of treatment withdrawal on negative affect
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Following drug treatment, the effects of morphine on negative affect (Fig. 2a) were evaluated during early or prolonged withdrawal in the FST (PND 45 and 70), NSF (PND 47 and 72) and
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SP (PND 63 and 83). In the FST, while no effect of Treatment was observed (F1,31=0.93, p=0.931), there was a significant effect of Sex (F1,31=5.05, p<0.05) and Day (F1,31=8.17, p<0.01). The effect of Day in male rats was only significant in those treated with morphine, presenting more immobility on PND 70 than on PND 45 (+6420 sec, p<0.01; Fig. 2a). In the NSF, there was a significant effect of Treatment (latency to feed: F1,31=5.53, p<0.05; feeding time: F1,31=5.39, p<0.05), Day (latency to feed: F1,31=23.15, p<0.001; feeding time:
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F1,31=97.95, p<0.001), Sex (latency to feed: F1,31=6.40, p<0.05) and Treatment x Day interaction (latency to feed: F8,256=7.80, p<0.01; feeding time: F1,31=7.45, p<0.05). Moreover, there was a significant Treatment x Day x Sex interaction when evaluating feeding time (F1,31=7.28, p<0.05). Morphine, as compared to saline-treated male rats, significantly decreased the latency to feed (-14637 sec, ***p<0.001) while increased the feeding time (+7113 sec, ***p<0.001) on PND 47, effects that were no longer observed on PND 72 (latency to feed: 5037 sec; feeding time: -113 sec; Fig. 2a). Moreover, the effect of Day (PND 72 vs. PND
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47) in male rats was only significant in those treated with morphine, presenting an increased latency to feed (+14927 sec, p<0.001) and a decreased feeding time (-9711 sec, p<0.001) on PND 72. Female rats, independently of treatment, showed decreased feeding time on PND
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72 vs. PND 47 (-7116 sec, p<0.05). Finally, in the SP, there was no effect of Treatment (F1,30=0.06, p=0.806) or Sex (F1,30=0.98, p=0.330), but a significant effect of Day (F1,30=4.48,
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p<0.05). The effect of Day in male rats was driven by rats treated with morphine that showed
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decreased SP by -8.52.1% on PND 83 vs. PND 63 (p<0.05; Fig. 2a).
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3.5. Effects of treatment withdrawal on NF protein content Morphine treatment during adolescence decreased the protein content of NF (NF-H-M: -
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187%, t=2.73, df=19, *p<0.05; NF-L: -269%, t=3.08, df=19, **p<0.01 vs. saline-treated rats) in the PFC of male rats during prolonged withdrawal (PND 89), while no effects were
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observed for female rats (Fig. 2b). Moreover, when evaluating through a two-way ANOVA the variable Sex for each protein evaluated, no overall significant differences were observed for NF-H-M (F1,31=0.496, p=0.487) or NF-L (F1,31=0.38, p=0.541).
4. Discussion
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This study evaluated the immediate and persistent behavioral and molecular consequences of adolescent morphine intake with a sex perspective. Basally, and prior to drug treatment, adolescent female rats showed smaller body weight and lower pain threshold than their male counterparts. Adolescent morphine treatment induced some sex-specific differences, while morphine impaired normal weight gain in male rats, no effects were observed for female rats. Plus, morphine produced an attenuated antinociceptive response in female rats. Moreover, following adolescent morphine treatment some emotional signs of withdrawal emerged
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exclusively in male rats in conjunction with signs of neurotoxicity. In particular, an anxiolyticlike effect was observed in adolescence during early withdrawal, which was followed by the development of a depressive-like phenotype (i.e., behavioral despair, anxiety-like behavior and
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anhedonia) in adulthood following prolonged withdrawal and paired with decreased contents of NF proteins in the PFC. Therefore, morphine administration during adolescence induced
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persistent changes in negative affect and brain toxicity selectively in male rats, suggesting
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female rats are resilient to these harmful effects.
Basally, and prior to drug treatment, adolescent female rats showed lower pain threshold than their male counterparts as observed by a smaller latency in the TF. In line with this, female
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rodents have shown lower pain threshold in experimental models of hot thermal 30,31. Interestingly, during adolescent morphine treatment, although no overall sex differences were
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observed (i.e., morphine was capable of inducing antinociception in both sexes), morphine
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exerted a reduced response in female rats, as observed by a smaller latency in the TF following 5 days of treatment (PND 42). This decreased antinociceptive effect induced by morphine in female rats, in terms of the magnitude of the analgesic response and potency, as compared to male rats has been long considered (e.g., 32). A big effort has been made to ascertain the mechanisms behind these sex-specific differences. A recent review summarized the existing
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literature regarding the neural basis for sex differences in opioid modulation of pain, with a focus on a sexually dimorphic descending opioid-induced inhibition 33. As expected, adolescent female rats showed smaller body weight than their male counterparts throughout the whole study. However, morphine treatment selectively impaired normal weight gain only in adolescent male rats, without affecting female rats, an effect that persisted, but did not worsen, throughout the course of withdrawal. Interestingly, cessation of morphine treatment has been associated with decreased body weight during withdrawal, an
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effect though to be mediated by the activation of stress-related brain circuits (e.g., 34,35), and whose magnitude was used as a predictive factor of drug withdrawal 36,37. Remarkably, most of these studies have utilized male rats. However, a prior study in Long Evans adult rats
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showed that while chronic morphine administration inhibited the typical weight gain in male rats, it potentiated the typical weight-gain pattern in female rats 38. Therefore, the present
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results extend the current literature by proving that adolescent morphine administration
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impaired body weight gain selectively in male rats, while failed to show the expected body weight drop typically observed during withdrawal for both sexes, in line with prior data
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suggesting that opioid withdrawal signs, regardless of sex, might be less severe in adolescent vs. adult rats (e.g., 10).
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When evaluating sex-specific emotional signs during morphine withdrawal, some effects emerged exclusively in male rats. Particularly, male rats showed improved anxiety-like
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behavior as measured 5 days post treatment on PND 47 in the NSF (i.e., decreased latency to feed and increased feeding time) when compared to saline-treated rats. In line with this response, one prior study also described mood elation during adolescent opioid withdrawal, observed as an antidepressant-like effect in male adolescent mice, while no effects were observed for female mice 16. However, given the higher motor activity previously described in response to morphine for adolescent male, but not female rats 39, one could not exclude
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the effect of locomotion over the enhanced response observed in male rats in the NSF. On the other hand, following prolonged withdrawal, male rats treated with morphine in adolescence showed increased negative affect as measured by increased behavioral despair (more immobility on PND 70 than on PND 45 in the FST), anxiety-like behavior (increased latency to feed and decreased feeding time on PND 72 as compared to PND 47 in the NSF) and anhedonia (decreased SP on PND 83 vs. PND 63). Notably, these effects were apparent in rats that received a challenge dose of morphine and were previously treated with morphine in
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adolescence (effect across time). Saline-treated rats during adolescence also received a single dose of morphine on PND 70, but no changes were observed in their response to the sequential tests as compared to their prior behavioral responses. These long-term effects emerging after
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the discontinuation of adolescent morphine administration, observed selectively in male rats, confirm that opiate withdrawal affect mood differently across sexes, being male rats more
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susceptible to develop a depressive-like phenotype in adulthood and thus more prone to relapse
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and addiction. Contrarily, prior literature showed that adult female rodents (and women) were the ones experiencing more negative adverse effects during withdrawal, and consequently more vulnerable than their male counterpart to relapse and to develop addiction 15. The present
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results suggest a different outcome when the drug experience occurs in adolescence, suggesting early initiation in drug consumption might change the outcome in terms of which sex shows a
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higher predisposition to the negative impact of morphine. Similarly, and in support of the
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present results, female rats were resistant to the long-lasting neurobehavioral changes induced by adolescent stress exposure 40. Furthermore, a recent study reported that male rats exhibited more severe withdrawal symptoms than females immediately following cessation of morphine administration 38. Therefore, the present results extend findings from earlier studies that indicated that male rats expressed more severe somatic opioid withdrawal than females 41,42. Thus, the emotional signs of withdrawal (e.g., anxiety, anhedonia, and
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aversion) emerging during adolescence seemed to be sex-specific and less intense than in their adult counterparts in line with previous data (e.g., 10). In terms of the possible brain markers that could accompany these sex-specific changes in behavior, the present study evaluated the content of NF proteins in the PFC. The results showed that following prolonged withdrawal male rats treated with morphine in adolescence showed decreased contents of NF proteins in the PFC, while no effects were observed in female rats. Since reduced levels of NF by opiates were associated with decreased axonal caliber and
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conduction velocity 43, impairment of axonal transport 44, possibly reflecting neural injury 20, the present results suggest certain degree of protection in female brains to the neurotoxic
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effect induced by adolescent morphine.
5. Conclusions
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While female rats were resilient to the negative effects of adolescent morphine on behavior and
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molecular changes, in male rats, morphine decreased adolescent body weight gain, and induced negative affect and brain toxicity during withdrawal. Given the widespread availability and use
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of opiate-based painkillers, the interplay between addiction, analgesia and emotional behaviors, and since adolescents and young adult humans are the age group with highest abuse
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potential, these results add to the current literature by reporting distinct sex-specific opioid actions when administered in adolescence. Caution should be taken when prescribing opioids
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in adolescents since the consequences of their intake are not completely well understood and might be different than expected during adulthood. Interestingly, in female rats, morphine was capable of inducing an analgesic response without any of the negative consequences observed in male rats.
Author contributions
F. Jiménez-Romero, C. Bis-Humbert, M.J. García-Fuster
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FJR and MJGF designed the study. FJR and CBH performed the behavioral testing, molecular experiments and undertook the statistical analysis. MJGF wrote the first draft of the manuscript. All authors contributed to and have approved the final version of the manuscript.
Role of Funding Source Funding for this study was provided by MSSSI Grant 2016/002 (Delegación del Gobierno para el Plan Nacional sobre Drogas, Ministerio de Sanidad, Servicios Sociales e Igualdad, Spain)
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and Fundación Alicia Koplowitz to MJG-F, while they had no further role in the: study design; collection, analysis and interpretation of data; writing of the report; and decision to submit the paper for publication. The program “TECH” from project “TALENT PLUS Construint Salut,
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Generant Valor" (Fundació Institut Investigació Sanitària Balears, Conselleria de Salut, GOIB)
Conflict of Interest
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Salud Carlos III, MINECO/FEDER).
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supports CB-H’s salary. MJG-F is a member of RETICS-RTA (RD16/0017/0010; Instituto de
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All authors declare that they have no conflicts of interest in relation to the work described.
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Pharmacol. Exp. Ther. 271, 1391-1398. 9 Grasing, K., Ghosh, S., 1998. Selegiline prevents long-term changes in dopamine efflux
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10 Radke, A.K., Gewirtz, J.C., Carroll, M.E., 2015. Effects of age, but not sex, on elevated startle during withdrawal from acute morphine in adolescent and adult rats. Behav. Pharmacol. 26, 485-488.
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11 Becker, J.B., Mazure, C.M., 2019. The federal plan for health science and technology’s response to the opioid crisis: understanding sex and gender differences as part of the solution is overlooked. Biol. Sex Diff. 10, 3. 12 Marsh, J.C., Park, K., Lin, Y.-A., Bersamira, C., 2018. Gender differences in trends for heroin use and nonmedical prescription opioid use, 2007–2014. J. Subst. Abuse Treat. 87, 79–85. 13 Greenfield, S.F., Brooks, A.J., Gordon, S.M., Green, C.A., Kropp, F., McHugh, R.K.,
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14 Becker, J.B., Koob, G.F., 2016. Sex differences in animal models: focus on addiction.
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15 Becker, J.B., Chartoff, E., 2019. Sex differences in neural mechanisms mediating reward
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17 Beitner‐Johnson, D., Guitart, X., Nestler, E.J., 1992. Neurofilament proteins and the
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mesolimbic dopamine system: common regulation by chronic morphine and chronic cocaine in the rat ventral tegmental area. J. Neurosci. 12, 2165–2176.
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18 Boronat, M.A., Olmos, G., García-Sevilla, J.A., 1998. Attenuation of tolerance to opioidinduced antinociception and protection against morphine-induced decrease of neurofilament proteins by idazoxan and other I2-imidazoline ligands. Br. J. Pharmacol. 125, 175-185.
19 García‐Sevilla, J.A., Ventayol, P., Busquets, X., La Harpe, R., Walzer, C., Guimon, J., 1997. Marked decrease of immunolabelled 68 kda neurofilament (Nf‐L) proteins in brains of opiate addicts. Neuro Report 8, 1561–1565.
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20 Nestler, E.J., Berhow, M.T., Brodkin, E.S., 1996. Molecular mechanism of drug addiction: adaptations in signal transduction pathways. Mol. Psychiat. 1, 190–199. 21 Jiménez Romero, F., Bis Humbert, C., García Fuster, M.J., 2019. Immediate and prolonged behavioural effects following chronic morphine administration during adolescence: A study focused on gender-based differences. Eur. Neuropsychopharmacol. 29, S483-S484. 22 McGrath, J.C., Lilley, E., 2015. Implementing guidelines on reporting research using
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animals (ARRIVE etc.): new requirements for publication in BJP. Br. J. Pharmacol. 172, 3189-3193.
23 García-Cabrerizo, R., Keller, B., García-Fuster, M.J., 2015. Hippocampal cell fate
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24 Turner, C.A., Gula, E.L., Taylor, L.P., Watson, S.J., Akil, H., 2008. Antidepressant-like effects of intracerebroventricular FGF2 in rats. Brain Res. 1224, 63-68.
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25 Spear, L.P., 2000. The adolescent brain and age-related behavioral manifestations. Neurosci. Biobehav. R. 24, 417–463.
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26 Boronat, M.A., García-Fuster, M.J., García-Sevilla, J.A., 2001. Chronic morphine induces up-regulation of the pro-apoptotic Fas receptor and down-regulation of the anti-apoptotic
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Bcl-2 oncoprotein in rat brain. Br. J. Pharmacol. 134, 1263-1270.
27 García-Cabrerizo, R., García-Fuster, M.J., 2019. Adolescent cocaine exposure enhanced negative affect following drug re-exposure in adult rats: Attenuation of c-Fos activation. J Psychopharmacol, 33:154-162.
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28 García-Cabrerizo, R., García-Fuster, M.J., 2019. Methamphetamine binge administration dose-dependently enhanced negative affect and voluntary drug consumption in rats following prolonged withdrawal: role of hippocampal FADD. Addict, Biol, 24, 239-250. 29 García-Cabrerizo, R., García-Fuster, M.J., 2015. Chronic MDMA induces neurochemical changes in the hippocampus of adolescent and young adult rats: Down-regulation of apoptotic markers. Neurotoxicology 49, 104-113. 30 Terner, J.M., Lomas, L.M., Smith, E.S., Barrett, A.C., Picker, M.J., 2003.
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Pharmacogenetic analysis of sex differences in opioid antinociception in rats. Pain 106, 381391.
31 Hurley, R.W., Adams, M.C., 2008. Sex, gender, and pain: an overview of a complex field.
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32 Kepler, K.L., Kest, B., Kiefel, J.M., Cooper, M.L., Bodnar, R.J., 1989. Roles of gender,
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gonadectomy and estrous phase in the analgesic effects of intracerebroventricular morphine
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33 Averitt, D.L., Eidson, L.N., Doyle, H.H., Murphy, A.Z., 2019. Neuronal and glial factors
155-165.
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contributing to sex differences in opioid modulation of pain. Neuropsychopharmacology 44,
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34 Maldonado, R., Fournié-Zaluski, M.C., Roques, B.P., 1992. Attenuation of the morphine withdrawal syndrome by inhibition of catabolism of endogenous enkephalins in the
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periaqueductal gray matter. Naunyn Schmiedebergs Arch. Pharmacol. 345, 466-472.
35 Houshyar, H., Manalo, S., Dallman, M.F., 2004. Time-dependent alterations in mRNA expression of brain neuropeptides regulating energy balance and hypothalamo-pituitaryadrenal activity after withdrawal from intermittent morphine treatment. J. Neurosci. 24, 9414-9424.
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36 Cicero, T.J., Meyer, E.R., 1973. Morphine pellet implantation in rats: quantitative assessment of tolerance and dependence. J. Pharmacol. Exp. Ther. 184, 404-408. 37 Gellert, V.F., Holtzman, S.G., 1978. Development and maintenance of morphine tolerance and dependence in the rat by scheduled access to morphine drinking solutions. J. Pharmacol. Exp. Ther. 205, 536-546. 38 Bobzean, S.A.M., Kokane, S.S., Butler, B.D., Perrotti, L.I., 2019. Sex differences in the
ventral tegmental area. Neurosci. Lett. 705, 124-130.
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expression of morphine withdrawal symptoms and associated activity in the tail of the
39 White, D.A., Michaels, C.C., Holtzman, S.G., 2008. Periadolescent male but not female
Biochemistry and Behavior 89, 188–199.
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rats have higher motor activity in response to morphine than do adult rats. Pharmacology
40 Klinger, K., Gomes, F.V., Rincón-Cortés, M., Grace, A.A., 2019. Female rats are resistant
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to the long-lasting neurobehavioral changes induced by adolescent stress exposure. Eur.
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Neuropsychopharmacol. doi: 10.1016/j.euroneuro.2019.07.134. 41 Craft, R.M., Stratmann, J.A., Bartok, R.E., Walpole, T.I., King, S.J., 1999. Sex differences
(Berl) 143, 1–7.
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in development of morphine tolerance and dependence in the rat. Psychopharmacology
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42 Cicero, T.J., Nock, B., Meyer, E.R., 2002. Gender-linked differences in the expression of physical dependence in the rat. Pharmacol. Biochem. Behav. 72, 691–697.
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43 Sakaguchi, T., Okada, M., Kitamura, T., Kawasaki, K., 1993. Reduced diameter and conduction velocity of myelinated fibers in the sciatic nerve of a neurofilament-deficient mutant quail. Neurosci. Lett. 153, 65-68.
44 Beitner‐Johnson, D., Nestler, E.J., 1993. Chronic morphine impairs mesolimbic transport in the rat mesolimbic dopamine system. NeuroReport 5, 57-60.
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Figure Legends Fig. 1 (a) Experimental design. FST: forced swim test; NSF: novelty-suppressed feeding test; PND: postnatal day; SP: sucrose preference test; PFC: prefrontal cortex; TF: tail-flick test; WB: western blot. (b) Basal measurements prior to drug treatment: body weight (g) across days (PND); behavioral despair as measured by time spent immobile (sec) in the FST on PND 28; anxiety-like behavior as measured by latency to feed (sec) in the NSF on PND 31; and antinociceptive response as measured by TF latency (sec) on PND 37. (c) Effects of morphine
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treatment on body weight (g) and (d) pain response (TF latency measured in sec). Data represents mean ± SEM of each measurement for each treatment group. Symbols represent individual rat values within each treatment group. Two- or three-way RM ANOVAs followed
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by Sidak's multiple comparisons tests or Student’s t-test were used for statistical analysis: (b) *p<0.05, ** p<0.01 and ***p<0.001 when compared to male rats; (c-d) *at least p<0.05 and
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p<0.05 when compared to male rats.
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***p<0.001 when compared to same-sex saline-treated rats at specified PND or # at least
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Fig. 2 (a) Effects of morphine withdrawal on negative affect: behavioral despair as measured by time spent immobile (sec) in the FST on PND 45 and 70; anxiety-like behavior as measured by latency to feed (sec) or feeding time (sec) in the NSF on PND 47 or 72; and
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hedonic-like response as measured by 1% SP in the two-bottle test on PND 63 or 83. Data represents mean ± SEM of each measurement for each treatment group. Three-way RM ANOVAs followed by Sidak's multiple comparisons tests: ***p<0.001 when compared to same-sex saline-treated rats at specified PND, at least <0.05 when comparing same-sex morphine-treated rats across PND, and # at least p<0.05 when compared to male rats.
(b) Effects of morphine withdrawal on NF protein content in the PFC. Data represents
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mean ± SEM of NF-H-M or NF-L protein content for each treatment group (expressed as % change vs. male-saline group). Symbols represent individual rat values within each treatment group. Student’s t-test were used for statistical analysis: *p<0.05 and **p<0.01 when compared
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to same-sex saline-treated rats. Representative immunoblots are shown depicting labeling of
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NF-H and NF-M, NF-L and -actin (as a loading control) for each set of experiments.
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PII:
S0304-3940(19)30728-1
DOI:
https://doi.org/10.1016/j.neulet.2019.134625
Reference:
NSL 134625
To appear in:
Neuroscience Letters
Received Date:
3 September 2019
Revised Date:
8 November 2019
Accepted Date:
9 November 2019
´ Please cite this article as: Jimenez-Romero F, Bis-Humbert C, Garc´ıa-Fuster MJ, Adolescent morphine induces emotional signs of withdrawal paired with neurotoxicity selectively in male rats: female resilience, Neuroscience Letters (2019), doi: https://doi.org/10.1016/j.neulet.2019.134625
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
F. Jiménez-Romero, C. Bis-Humbert, M.J. García-Fuster
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Research Article NSL-191417-R1
Adolescent morphine induces emotional signs of withdrawal paired with neurotoxicity selectively in male rats: female resilience
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Fernando Jiménez-Romero, Cristian Bis-Humbert, M. Julia García-Fuster IUNICS, University of the Balearic Islands, and IdISBa, Palma, Spain
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Short title: Sex-specific effects of adolescent morphine in rats
Correspondence to: Dr. M. Julia García-Fuster. IUNICS, University of the Balearic Islands, Cra. de Valldemossa km 7.5, E-07122 Palma, Spain.
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Phone: +34 971 259992 Email: [email protected]
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Highlights
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Fax: +34 971 259501
Adolescent morphine induced persistent sex-dependent changes in negative affect.
Adolescent morphine induced persistent sex-dependent changes in brain toxicity.
Female rats seemed resilient to the negative effects induced by adolescent morphine.
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Abstract This study evaluated the immediate and persistent behavioral and molecular consequences (i.e., emotional signs emerging during withdrawal and signs of neurotoxicity) following adolescent
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morphine exposure with a sex perspective. Basally, and prior to drug treatment, adolescent female rats showed smaller body weight and lower pain threshold than their male counterparts. Adolescent morphine treatment induced some sex-specific differences, while morphine impaired normal weight gain in male rats, no effects were observed for female rats. Plus, morphine produced an attenuated antinociceptive response in female rats. Moreover, following adolescent morphine treatment some emotional signs of withdrawal emerged exclusively in male rats in conjunction with signs of neurotoxicity, while female rats were not affected. In
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particular, an anxiolytic-like effect in adolescence during early withdrawal was followed by the development of a depressive-like phenotype (i.e., behavioral despair, anxiety-like behavior and anhedonia) in adulthood during prolonged withdrawal and paired with decreased contents
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of neurofilaments proteins in the prefrontal cortex. In conclusion, morphine administration during adolescence induced persistent changes in negative affect and brain toxicity selectively
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in male rats, suggesting female rats were resilient to these harmful effects. Given the
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widespread availability and use of opiate-based painkillers, the interplay between addiction, analgesia and emotional behaviors, and since adolescents and young adult humans are the age group with the highest abuse potential, these results add to the current literature by reporting
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Keywords
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distinct sex-specific opioid actions when administered in adolescence.
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Sex differences; morphine; adolescence; withdrawal; age.
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1. Introduction Opioids accounted for 70% of the negative health impact associated with drug use disorders worldwide 1, being adolescents and young adult humans aged 12-25 years old more likely than any other age group to abuse illegal opioids or misuse prescription pain relievers 2. Studies in adolescent rodent models suggest that the response to opiates changes during development (e.g., 3). For example, adolescent rats self-administer less morphine than adults, an effect that was interpreted as higher drug sensitivity, and show lower rates of reinstatement,
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suggesting adolescent development might include some protective factors that diminish the long-term impact of early drug intake 4. This seems particularly relevant given that an early onset of opioids use has been associated with an increased chance of having a physical or
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psychiatric co-morbidity 5. In this context, opioid withdrawal in humans and rodents is accompanied by the precipitation of negative affect, such as anxiety, anhedonia, and aversion
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6-9. While these emotional signs of withdrawal tend to be experienced earlier and less
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severely in adolescent rodents than in their adult counterparts (e.g. 10), these studies have been mainly centered in the alcohol and nicotine fields. Therefore, age-related differences in
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affective responses to morphine withdrawal might explain the increased risk of mental illness when drug use begins during adolescence.
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Sex must be also considered to understand opioid use disorder (see commentary in 11). For instance, studies in humans and rodents suggest that females may be at greater risk for the
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nonmedical use of prescription opioids than for the use of heroin 12, while also appear to shift faster from a casual use of opioids to dependence 13,14, and show higher levels of craving and relapse during abstinence (see revision in 15), suggesting sex-specific factors for addiction liability related to differences in opioid signaling in the brain. Opiate withdrawal might affect mood differentially across sexes (e.g., 16,15). In this sense, sex differences in affective responses to withdrawal might contribute to the differential development of comorbid
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mental disorders in female and male rodents. However, there is a lack of studies on sex differences in affective-like responses to opioid withdrawal, especially during adolescence. Thus, the first aim of this study was to determine sex-based differences in the emotional signs of withdrawal that emerge following an escalating repeated morphine regimen in rats during adolescence. To date, the molecular mechanisms underlying morphine withdrawal have been mainly studied in male adult animals. In this context, prolonged opiate exposure is known to induce
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marked decreases in brain cytoeskeletal proteins, such as decreases in neurofilament (NF) proteins in the prefrontal cortex (PFC) of rats 17,18 and of opiate addicts 19, suggesting that such plastic changes may reflect the induction of cortical neuronal damage 20. However,
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when looking more into the specifics of these studies and of the prior literature, the reports have been centered in males, for example in 19, 16 out of 17 human subjects were males and
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only 1 female was included in the study. Moreover, and to be the best of our knowledge, no
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prior experiments evaluated the effects of morphine on NF protein content in female rodents or following adolescent morphine exposure. In this context, the second aim of this study was
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to determine sex-specific neurotoxicity induced by adolescent exposure that could accompany the sex-specific changes in emotional behavior during drug withdrawal. A preliminary report of this work was presented at the 31st European College of Neuropsychopharmacology
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Congress 21.
2. Material and methods 2.1. Animals
A total of 36 Sprague-Dawley rats (22 male and 14 female) from 3 litters bred at the same time in the animal facility at the University of the Balearic Islands were used. Rats were housed in standard cages (groups of 2 to 3) under precise environmental conditions such as 22 °C, 70%
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humidity, and 12 h light/dark cycle (lights on at 8:00 AM) and with ad libitum access to a standard diet and tap water. Animal experiments complied with ARRIVE guidelines 22 and EC Directive 86/609/EEC. All experimental procedures were performed during the light period and were previously approved by the Spanish Royal Decree 53/2013, the Local Bioethical Committee (University of the Balearic Islands) and the regional Government (Conselleria Medi Ambient, Agricultura i Pesca, Direcció General Agricultura i Ramaderia, Govern de les Illes
2.2. Behavioral testing and pharmacological treatment
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Balears).
Emotional state measures (i.e., core symptoms of a depressive-like phenotype) were evaluated
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by 3 consecutive experimental tests, forced-swim test (FST), novelty suppressed feeding (NSF) and sucrose preference (SP) that measure changes in behavioral despair, anxiety-like behavior
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and hedonic-like responses respectively. After weaning and prior to any drug treatment, some
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basal measurements were taken (see details in Fig. 1a). Rats were weighted on postnatal days (PND) 26, 29, 31 and 35. They were exposed to the stress of the FST, which consisted of a 15 min pre-test on PND 27 followed by a 5 min test on PND 28 (this session was videotaped).
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Briefly, each rat was individually placed in a tank (41 cm high x 32 cm diameter), with water to a depth of 25 cm, and at 25 1 C as described before in 23. Videos were blinded for
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analysis to score individual performances in the basal time spent immobile (sec) (Behavioral
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Tracker software, CA, USA). Then, rats were exposed to the NSF (see 24). To do so, rats were food deprived for 48 h (starting on PND 29) prior to testing on PND 31 in an open-field apparatus (square arena: 60 x 60 cm and 40 cm of height) with a small piece of rat chow placed in the center of the arena. Each animal started in a corner of the arena and was allowed a maximum of 5 min to begin feeding. Sessions were recorded and videos were analyzed blind to the experimental groups using a digital video tracking system (Smart Video Tracking
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software, Version 3.0.03, Panlab SL, Barcelona, Spain) to calculate the latency to feed (sec) as well as the feeding time (sec). After testing, animals were placed in their home cage with food and water ad libitum. Finally, the tail-flick test (TF) was performed on PND 37 as described earlier 18 to measure basal latencies for each rat (i.e. average of 3 individual measurements for each rat) one day prior to treatment. The TF latency was defined as the time from the onset of radiant heat to tail withdrawal. To minimize tail skin tissue damage, cutoff time was set as 3 times the baseline latency.
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Then, rats were repeatedly treated during mid-adolescence (3 times/day, for 4 days, from PND 38-41 and 2 times on PND 42, spaced 5-6 hours during the lights on period, i.p.; see revision in 25 for developmental stages for rodents) with saline (0.9% NaCl, 1 ml/kg, n=11
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for the male group and n=7 for the female group) or with an escalating regimen (10-100 mg/kg, n=11 for the male group and n=7 for the female group) of morphine HCl (Unión Químico-
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Farmacéutica S.A.E., Madrid, Spain). The daily doses of morphine were based on prior studies
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from our research group (e.g., 26): day 1, 10/10/10 mg/kg; day 2, 10/20/20 mg/kg; day 3, 20/20/40 mg/kg; day 4, 40/40/80 mg/kg; and day 5, 80/100 mg/kg. The TF was used to evaluate
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the antinociceptive response of morphine on PND 38, 40 and 42 (days 1, 3 and 5 of treatment respectively) as measured 1 h after the first daily injection. Consecutive behavioral tests were
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then performed to evaluate negative affect during immediate (PND 45-64) and prolonged withdrawal (PND 70-84, following an analgesic challenge dose of 10 mg/kg of morphine
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administered to all 36 rats to compare the acute effects of morphine in naïve rats vs. its effects in the FST in rats previously exposed to morphine in adolescence on PND 70): (1) FST to evaluate behavioral despair (a 5 min test on PND 45 and PND 70); (2) NSF for anxiety-like behavior (a 5 min test on PND 47 and PND 72 following food removal 48 h prior to test); and (3) 1% SP (two bottle choice) test for hedonic-like response (a 48 h test on PND 62-63 and PND 82-83) (see further details in 27,28). Rats were single housed starting on PND 56 (to
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evaluate the individual hedonic response) and remained like that until the end of the experiment. This experimental design was set to evaluate the temporal course of the behavioral effects emerging during morphine withdrawal. To avoid the effects of learning and/or the development of tolerance to the test performance due to repetition of the behavioral tests, the same conditions were followed for all rats, in an attempt to compare the progression of treated rats vs. their respective controls (see 27 for similar procedures).
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2.3. Tissue collection for western blot analysis
Rats were sacrificed on PND 89 (Fig. 1a), the PFC was freshly dissected, immediately frozen in liquid nitrogen and stored at -80 C until tissue was prepared for western blot analysis
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following standardized lab protocols: 40 μg of total protein for each sample were resolved by electrophoresis, transferred to a nitrocellulose membrane and incubated with selected
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antibodies (e.g., 29). Information about primary antibodies, previously characterized by our
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group 18,19,29, regarding vendors and dilution conditions: (1) Covance (CA, USA): antiNF-H and M (clone SMI-32; 1:1000); and (2) Sigma-Aldrich (MO, USA): anti-NF-L (N5139;
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1:1000), anti-ß-actin (clone AC-15; 1:10000). The corresponding secondary antibodies (antirabbit or anti-mouse IgG linked to horseradish peroxidase) were incubated for 1 h at room
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temperature (1:5000 dilution; Cell Signaling). Immunoreactivity of target proteins was detected with ECL reagents (Amersham, Buckinghamshire, UK) and signal of bound antibody
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was visualized by exposure to autoradiographic film (Amersham ECL Hyperfilm) for 1 to 60 min, which was quantified by densitometric scanning (GS-800 Imaging Calibrated Densitometer, Bio-Rad). Percent changes in immunoreactivity were calculated for each rat in each gel with respect to male control samples (M-Sal: 100%). Each rat sample was run at least 2-3 times in different gels, and the mean value was used as a final estimate. The content of ßactin was used as loading control.
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2.4. Statistical analyses Data were analyzed with GraphPad Prism, Version 8 (GraphPad Software, Inc.). Results are expressed as mean values ± standard error of the mean (SEM). Two (Sex and Day) or threeway (Treatment, Sex and Day) ANOVAs (with or without repeated measures, RM) followed by Sidak's multiple comparisons test when appropriate or two-tail Student's t-tests (for changes
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in NF content) were used as statistical analyses. The level of significance was set at p≤0.05.
3. Results 3.1. Basal measurements
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As expected, there were basal body weight differences in male and female offspring (Sex x Day interaction: F3,102=10.89, p<0.001; Fig. 1b). In particular, female rats weighted less than
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their male siblings at any time point evaluated (*p<0.05; **p<0.01 and ***p<0.001). When
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evaluating behavioral despair (time spent immobile) in the FST (PND 28), no basal differences between male and female rats were observed (t=1.46, df=34, p=0.152; Fig. 1b). Similarly, no basal changes were observed for anxiety-like behavior (latency to feed in the NSF; t=0.41,
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df=34, p=0.684; Fig. 1b) on PND 31. However, when basal pain response was evaluated in the TF on PND 37, female rats showed a reduced latency to remove the tail from the hot laser
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exposure (-0.970.33 sec vs. male rats; t=2.89, df=34, **p<0.01; Fig. 1b).
3.2. Effects of treatment on body weight As shown in Fig. 1c, when measuring the effects of treatment on body weight there was a significant effect of Treatment (F1,32=8.91, p<0.01), Sex (F1,32=215.4, p<0.001), Day
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(F1.742,55.73=2863, p<0.001) and Sex x Day interaction (F8,256=293.9, p<0.001). In particular, when evaluating the effect of Treatment male rats treated with morphine showed reduced normal weight gain following 4 days of treatment (PND 41; -237.5 g, *p<0.05 vs. salinetreated rats), an effect that remained significant throughout the rest of the study (from PND 4780, range of smaller body weight from -20 to -247.5 g, *p<0.05, Fig. 1c). Female rats continued to be smaller than their male counterparts and showed no changes by Treatment.
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3.3. Effects of treatment on antinociception
During drug treatment (PND 28-42), the antinociceptive effects of morphine were evaluated in the TF test on PND 38, 40 and 42 (days 1, 3 and 5 of treatment respectively). There was a
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significant effect of Treatment (F1,32=166.4, p<0.001) and Sex (F1,32=3.99, p=0.05), but not of Day (F2,64=0.92, p=0.404). In particular, although the effect of Treatment was observed for
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male and female rats (i.e., increased TF latency by morphine vs. saline-treated rats;
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***p<0.001; Fig. 1d) at all days measured, female rats showed a smaller increase induced by
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morphine (e.g., -1.55 0.49 sec on PND 42 vs. male morphine-treated rats, #p < 0.01, Fig. 1d).
3.4. Effects of treatment withdrawal on negative affect
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Following drug treatment, the effects of morphine on negative affect (Fig. 2a) were evaluated during early or prolonged withdrawal in the FST (PND 45 and 70), NSF (PND 47 and 72) and
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SP (PND 63 and 83). In the FST, while no effect of Treatment was observed (F1,31=0.93, p=0.931), there was a significant effect of Sex (F1,31=5.05, p<0.05) and Day (F1,31=8.17, p<0.01). The effect of Day in male rats was only significant in those treated with morphine, presenting more immobility on PND 70 than on PND 45 (+6420 sec, p<0.01; Fig. 2a). In the NSF, there was a significant effect of Treatment (latency to feed: F1,31=5.53, p<0.05; feeding time: F1,31=5.39, p<0.05), Day (latency to feed: F1,31=23.15, p<0.001; feeding time:
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F1,31=97.95, p<0.001), Sex (latency to feed: F1,31=6.40, p<0.05) and Treatment x Day interaction (latency to feed: F8,256=7.80, p<0.01; feeding time: F1,31=7.45, p<0.05). Moreover, there was a significant Treatment x Day x Sex interaction when evaluating feeding time (F1,31=7.28, p<0.05). Morphine, as compared to saline-treated male rats, significantly decreased the latency to feed (-14637 sec, ***p<0.001) while increased the feeding time (+7113 sec, ***p<0.001) on PND 47, effects that were no longer observed on PND 72 (latency to feed: 5037 sec; feeding time: -113 sec; Fig. 2a). Moreover, the effect of Day (PND 72 vs. PND
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47) in male rats was only significant in those treated with morphine, presenting an increased latency to feed (+14927 sec, p<0.001) and a decreased feeding time (-9711 sec, p<0.001) on PND 72. Female rats, independently of treatment, showed decreased feeding time on PND
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72 vs. PND 47 (-7116 sec, p<0.05). Finally, in the SP, there was no effect of Treatment (F1,30=0.06, p=0.806) or Sex (F1,30=0.98, p=0.330), but a significant effect of Day (F1,30=4.48,
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p<0.05). The effect of Day in male rats was driven by rats treated with morphine that showed
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decreased SP by -8.52.1% on PND 83 vs. PND 63 (p<0.05; Fig. 2a).
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3.5. Effects of treatment withdrawal on NF protein content Morphine treatment during adolescence decreased the protein content of NF (NF-H-M: -
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187%, t=2.73, df=19, *p<0.05; NF-L: -269%, t=3.08, df=19, **p<0.01 vs. saline-treated rats) in the PFC of male rats during prolonged withdrawal (PND 89), while no effects were
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observed for female rats (Fig. 2b). Moreover, when evaluating through a two-way ANOVA the variable Sex for each protein evaluated, no overall significant differences were observed for NF-H-M (F1,31=0.496, p=0.487) or NF-L (F1,31=0.38, p=0.541).
4. Discussion
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This study evaluated the immediate and persistent behavioral and molecular consequences of adolescent morphine intake with a sex perspective. Basally, and prior to drug treatment, adolescent female rats showed smaller body weight and lower pain threshold than their male counterparts. Adolescent morphine treatment induced some sex-specific differences, while morphine impaired normal weight gain in male rats, no effects were observed for female rats. Plus, morphine produced an attenuated antinociceptive response in female rats. Moreover, following adolescent morphine treatment some emotional signs of withdrawal emerged
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exclusively in male rats in conjunction with signs of neurotoxicity. In particular, an anxiolyticlike effect was observed in adolescence during early withdrawal, which was followed by the development of a depressive-like phenotype (i.e., behavioral despair, anxiety-like behavior and
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anhedonia) in adulthood following prolonged withdrawal and paired with decreased contents of NF proteins in the PFC. Therefore, morphine administration during adolescence induced
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persistent changes in negative affect and brain toxicity selectively in male rats, suggesting
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female rats are resilient to these harmful effects.
Basally, and prior to drug treatment, adolescent female rats showed lower pain threshold than their male counterparts as observed by a smaller latency in the TF. In line with this, female
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rodents have shown lower pain threshold in experimental models of hot thermal 30,31. Interestingly, during adolescent morphine treatment, although no overall sex differences were
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observed (i.e., morphine was capable of inducing antinociception in both sexes), morphine
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exerted a reduced response in female rats, as observed by a smaller latency in the TF following 5 days of treatment (PND 42). This decreased antinociceptive effect induced by morphine in female rats, in terms of the magnitude of the analgesic response and potency, as compared to male rats has been long considered (e.g., 32). A big effort has been made to ascertain the mechanisms behind these sex-specific differences. A recent review summarized the existing
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literature regarding the neural basis for sex differences in opioid modulation of pain, with a focus on a sexually dimorphic descending opioid-induced inhibition 33. As expected, adolescent female rats showed smaller body weight than their male counterparts throughout the whole study. However, morphine treatment selectively impaired normal weight gain only in adolescent male rats, without affecting female rats, an effect that persisted, but did not worsen, throughout the course of withdrawal. Interestingly, cessation of morphine treatment has been associated with decreased body weight during withdrawal, an
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effect though to be mediated by the activation of stress-related brain circuits (e.g., 34,35), and whose magnitude was used as a predictive factor of drug withdrawal 36,37. Remarkably, most of these studies have utilized male rats. However, a prior study in Long Evans adult rats
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showed that while chronic morphine administration inhibited the typical weight gain in male rats, it potentiated the typical weight-gain pattern in female rats 38. Therefore, the present
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results extend the current literature by proving that adolescent morphine administration
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impaired body weight gain selectively in male rats, while failed to show the expected body weight drop typically observed during withdrawal for both sexes, in line with prior data
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suggesting that opioid withdrawal signs, regardless of sex, might be less severe in adolescent vs. adult rats (e.g., 10).
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When evaluating sex-specific emotional signs during morphine withdrawal, some effects emerged exclusively in male rats. Particularly, male rats showed improved anxiety-like
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behavior as measured 5 days post treatment on PND 47 in the NSF (i.e., decreased latency to feed and increased feeding time) when compared to saline-treated rats. In line with this response, one prior study also described mood elation during adolescent opioid withdrawal, observed as an antidepressant-like effect in male adolescent mice, while no effects were observed for female mice 16. However, given the higher motor activity previously described in response to morphine for adolescent male, but not female rats 39, one could not exclude
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the effect of locomotion over the enhanced response observed in male rats in the NSF. On the other hand, following prolonged withdrawal, male rats treated with morphine in adolescence showed increased negative affect as measured by increased behavioral despair (more immobility on PND 70 than on PND 45 in the FST), anxiety-like behavior (increased latency to feed and decreased feeding time on PND 72 as compared to PND 47 in the NSF) and anhedonia (decreased SP on PND 83 vs. PND 63). Notably, these effects were apparent in rats that received a challenge dose of morphine and were previously treated with morphine in
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adolescence (effect across time). Saline-treated rats during adolescence also received a single dose of morphine on PND 70, but no changes were observed in their response to the sequential tests as compared to their prior behavioral responses. These long-term effects emerging after
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the discontinuation of adolescent morphine administration, observed selectively in male rats, confirm that opiate withdrawal affect mood differently across sexes, being male rats more
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susceptible to develop a depressive-like phenotype in adulthood and thus more prone to relapse
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and addiction. Contrarily, prior literature showed that adult female rodents (and women) were the ones experiencing more negative adverse effects during withdrawal, and consequently more vulnerable than their male counterpart to relapse and to develop addiction 15. The present
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results suggest a different outcome when the drug experience occurs in adolescence, suggesting early initiation in drug consumption might change the outcome in terms of which sex shows a
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higher predisposition to the negative impact of morphine. Similarly, and in support of the
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present results, female rats were resistant to the long-lasting neurobehavioral changes induced by adolescent stress exposure 40. Furthermore, a recent study reported that male rats exhibited more severe withdrawal symptoms than females immediately following cessation of morphine administration 38. Therefore, the present results extend findings from earlier studies that indicated that male rats expressed more severe somatic opioid withdrawal than females 41,42. Thus, the emotional signs of withdrawal (e.g., anxiety, anhedonia, and
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aversion) emerging during adolescence seemed to be sex-specific and less intense than in their adult counterparts in line with previous data (e.g., 10). In terms of the possible brain markers that could accompany these sex-specific changes in behavior, the present study evaluated the content of NF proteins in the PFC. The results showed that following prolonged withdrawal male rats treated with morphine in adolescence showed decreased contents of NF proteins in the PFC, while no effects were observed in female rats. Since reduced levels of NF by opiates were associated with decreased axonal caliber and
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conduction velocity 43, impairment of axonal transport 44, possibly reflecting neural injury 20, the present results suggest certain degree of protection in female brains to the neurotoxic
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effect induced by adolescent morphine.
5. Conclusions
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While female rats were resilient to the negative effects of adolescent morphine on behavior and
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molecular changes, in male rats, morphine decreased adolescent body weight gain, and induced negative affect and brain toxicity during withdrawal. Given the widespread availability and use
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of opiate-based painkillers, the interplay between addiction, analgesia and emotional behaviors, and since adolescents and young adult humans are the age group with highest abuse
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potential, these results add to the current literature by reporting distinct sex-specific opioid actions when administered in adolescence. Caution should be taken when prescribing opioids
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in adolescents since the consequences of their intake are not completely well understood and might be different than expected during adulthood. Interestingly, in female rats, morphine was capable of inducing an analgesic response without any of the negative consequences observed in male rats.
Author contributions
F. Jiménez-Romero, C. Bis-Humbert, M.J. García-Fuster
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FJR and MJGF designed the study. FJR and CBH performed the behavioral testing, molecular experiments and undertook the statistical analysis. MJGF wrote the first draft of the manuscript. All authors contributed to and have approved the final version of the manuscript.
Role of Funding Source Funding for this study was provided by MSSSI Grant 2016/002 (Delegación del Gobierno para el Plan Nacional sobre Drogas, Ministerio de Sanidad, Servicios Sociales e Igualdad, Spain)
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and Fundación Alicia Koplowitz to MJG-F, while they had no further role in the: study design; collection, analysis and interpretation of data; writing of the report; and decision to submit the paper for publication. The program “TECH” from project “TALENT PLUS Construint Salut,
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Generant Valor" (Fundació Institut Investigació Sanitària Balears, Conselleria de Salut, GOIB)
Conflict of Interest
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Salud Carlos III, MINECO/FEDER).
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supports CB-H’s salary. MJG-F is a member of RETICS-RTA (RD16/0017/0010; Instituto de
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All authors declare that they have no conflicts of interest in relation to the work described.
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Figure Legends Fig. 1 (a) Experimental design. FST: forced swim test; NSF: novelty-suppressed feeding test; PND: postnatal day; SP: sucrose preference test; PFC: prefrontal cortex; TF: tail-flick test; WB: western blot. (b) Basal measurements prior to drug treatment: body weight (g) across days (PND); behavioral despair as measured by time spent immobile (sec) in the FST on PND 28; anxiety-like behavior as measured by latency to feed (sec) in the NSF on PND 31; and antinociceptive response as measured by TF latency (sec) on PND 37. (c) Effects of morphine
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treatment on body weight (g) and (d) pain response (TF latency measured in sec). Data represents mean ± SEM of each measurement for each treatment group. Symbols represent individual rat values within each treatment group. Two- or three-way RM ANOVAs followed
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by Sidak's multiple comparisons tests or Student’s t-test were used for statistical analysis: (b) *p<0.05, ** p<0.01 and ***p<0.001 when compared to male rats; (c-d) *at least p<0.05 and
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p<0.05 when compared to male rats.
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***p<0.001 when compared to same-sex saline-treated rats at specified PND or # at least
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Fig. 2 (a) Effects of morphine withdrawal on negative affect: behavioral despair as measured by time spent immobile (sec) in the FST on PND 45 and 70; anxiety-like behavior as measured by latency to feed (sec) or feeding time (sec) in the NSF on PND 47 or 72; and
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hedonic-like response as measured by 1% SP in the two-bottle test on PND 63 or 83. Data represents mean ± SEM of each measurement for each treatment group. Three-way RM ANOVAs followed by Sidak's multiple comparisons tests: ***p<0.001 when compared to same-sex saline-treated rats at specified PND, at least <0.05 when comparing same-sex morphine-treated rats across PND, and # at least p<0.05 when compared to male rats.
(b) Effects of morphine withdrawal on NF protein content in the PFC. Data represents
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mean ± SEM of NF-H-M or NF-L protein content for each treatment group (expressed as % change vs. male-saline group). Symbols represent individual rat values within each treatment group. Student’s t-test were used for statistical analysis: *p<0.05 and **p<0.01 when compared
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to same-sex saline-treated rats. Representative immunoblots are shown depicting labeling of
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NF-H and NF-M, NF-L and -actin (as a loading control) for each set of experiments.
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