Incubation of social deficit during morphine abstinence in male mice using a novel unbiased and automatized method

Abstract

Introduction: Opioid use disorder (OUD) is a chronic relapsing condition caused by prolonged opioid exposure, which triggers adaptive changes in the brain. These changes make it challenging to control or abstain from consuming, and significantly increase the risk of relapse. While the physical symptoms of withdrawal typically resolve within a few days, extended abstinence is frequently accompanied by the progressive development of emotional disturbances. Additionally, abstinent individuals often report social disengagement, or even social isolation that worsen the condition and participates in the development of comorbidities. These disturbances are similarly observed in murine models of opioid abstinence.

Methods: However, traditional methods for assessing social deficits in rodents often rely on simplistic paradigms with limited behavioral metrics. Here, we utilized a well-established model of morphine administration followed by protracted abstinence, combined with the Live Mouse Tracker (LMT) system. Using the real-time video-based automated LMT system, we conducted longitudinal recordings of social behaviors over a 4-week period of morphine abstinence, during repeated social interaction sessions.

Results: The use of this method, offering an unbiased and precise behavioral characterization of social investigation between freely-moving male mice, revealed that while motor and activity-related disruptions emerge and resolve quickly immediately following the onset of abstinence, social deficits progressively intensify over time, reaching their peak 3 weeks after the final morphine administration. Additionally, the LMT provided detailed insights into subtle behavioral changes throughout the course of abstinence and within individual but also that early deficits in explorations and social interactions might serve as predictor for the severity of the late social deficits.

Discussion: These results point out the need to improve and implement unbiased tracking methods for a deeper and refined understanding of rodent behaviors modeling psychiatric conditions.

Keywords: morphine; opioid; protracted abstinence; social deficits; withdrawal.

Figure 1

Morphine withdrawal induced a decrease of spontaneous exploration in absence of unfamiliar mouse. (a) Graphical representation of the behavioral design. Mice were recorded for 15 min (habituation) in an empty open field (OF) and then during a 25 min session of direct social interaction. Following a baseline (BL) session half of the mice received an escalating morphine treatment and then recorded in the same OF paradigm 24 h, one, two, three and for weeks post-treatment. (b) Screenshot of the LMT viewer [from de Chaumont et al. (2019)]. (c) Example of two LMT reconstructions, during habituation (left panel) and during social interaction (right panel). (d) Mean weight loss before and after the last day of treatment. (e) Mean distance traveled during the habituation phase for morphine- (brown) and saline-treated (grey) mice, across sessions. (f) Mean distance traveled in the center of the OF during the habituation phase for morphine- and saline-treated mice, across sessions. (g) Mean time spent in the center of the OF during the habituation phase for morphine- and saline-treated mice, across sessions. (h) Mean number of rearing during the habituation phase for morphine- and saline-treated mice, across sessions. (i) Mean rearing duration during the habituation phase for morphine- and saline-treated mice, across sessions. (j) Mean average rearing duration during the habituation phase for morphine- and saline-treated mice, across sessions. Data are represented as mean ± SEM. Morphine n = 11 and Saline control n = 10. *p < 0.05; ****p < 0.0001 for the comparison between saline- and morphine-treated mice.

Figure 2

Protracted morphine decreased social induced activity behaviors differentially over time. (a) Graphical representation of the behavioral design. (b) Mean distance traveled during the interaction phase for morphine- (brown) and saline-treated (grey) mice, across sessions. (c) Mean number of total moves (animal moving at a speed >5 cm/s) during the interaction phase for morphine- and saline-treated mice, across sessions. (d) Mean number of total moves while isolated during the interaction phase for morphine- and saline-treated mice, across sessions. (e) Mean number of total moves in contact with the interactor during the interaction phase for morphine- and saline-treated mice, across sessions. Data are represented as mean ± SEM. Morphine n = 11 and Saline control n = 10. *p<0.05; **p<0.01 for the comparison between saline- and morphine-treated mice.

Figure 3

Incubation of the social deficit induced by morphine protracted abstinence. (a) Mean number of social approaches during the interaction phase for morphine- (brown) and saline-treated (grey) mice, across sessions. (b) Mean number of contacts during the interaction phase for morphine- and saline-treated mice, across sessions. (c) Mean number of dyadic events during the interaction phase for morphine- and saline-treated mice, across sessions. (d) Mean number of dyadic state events during the interaction phase for morphine- and saline-treated mice, across sessions. (e) Mean number of dyadic dynamic events during the interaction phase for morphine- and saline-treated mice, across sessions. Data are represented as mean ± SEM. Morphine n = 11 and Saline control n = 10. *p < 0.05 for the comparison between saline- and morphine-treated mice.

Figure 4

Protracted morphine decreased social interaction at a late stage but not early. (a) Mean Z-score (normalized to the saline mice) for the contacts for morphine- (brown) and saline-treated (grey) mice, at the early (24 h to 1 week) and late (2 to 4 weeks) stages. (b) Z-score (normalized to the saline mice) for the social approaches for morphine- and salinetreated mice, at the early and late stages. (c) Mean Z-score (normalized to the saline mice) for the dyadic events for morphine- and saline-treated mice, at the early and late stages. (d) Mean combined Z-score (normalized to the saline mice) for morphine- and saline-treated mice, at the early and late stages. This Z-score was obtained by averaging each individual Z-score of the number of contacts, social approaches and dyadic events. Data are represented as mean ± SEM. Morphine n = 11 and Saline control n = 10. *p < 0.05 for the comparison between saline- and morphine-treated mice; &&p < 0.01 for the comparison between the early and late phase for the morphine-treated mice.

Figure 5

Early motor deficits predict social deficits induced by morphine protracted abstinence. (a) Mean Z-score for the “motor” variables analyzed during the OF habituation sessions for morphine- (brown) and saline-treated (grey) mice. Data are represented as mean ± SEM. ***p < 0.001 for the comparison between saline- and morphine-treated mice; &&&&p < 0.0001 for the comparison between the early and late phase for the morphine-treated mice. (b) Linear regression showing a positive correlation between the early motor deficits (motor Z-score for the early phase) and the social Z-score at late (r² = 0.33, p = 0.007; red circles) but not early stage (r² = 0.06, p = 0.29; white circles). (c) Linear regression demonstrating the positive correlation between the social Z-scores at early and late stages (r² = 0.35, p = 0.005). (d) Linear regression showing a positive correlation between the weight loss during morphine treatment and the social Z-score at late (r² = 0.26, p = 0.02; red circles) but not early stage (r² = 0.01, p = 0.66; white circles). Morphine n = 11 and Saline control n = 10.