# Model of negative affect induced by withdrawal from acute and chronic morphine administration in male mice

Dersu Ozdemir, Judith Meyer, Brigitte L Kieffer, Emmanuel Darcq

► To cite this version:

Dersu Ozdemir, Judith Meyer, Brigitte L Kieffer, Emmanuel Darcq. Model of negative affect induced by withdrawal from acute and chronic morphine administration in male mice. Scientific Reports, 2024, 14 (1), pp.9767. 10.1038/s41598-024-60759-3 . inserm-05509541

HAL Id: inserm-05509541

https://inserm.hal.science/inserm-05509541v1

Submitted on 13 Feb 2026

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
<figure><img src="image_2.png" /></figure>

# Model of negative affect induced by withdrawal from acute and chronic morphine administration in male mice

Dersu Ozdemir<fnref n="1" />, Judith Meyer<fnref n="1" />, Brigitte L. Kieffer<fnref n="1" />,² & Emmanuel Darcq<fnref n="1" />,²✉

Opioid use disorder (OUD) is a chronic relapsing disorder that is a major burden for the lives of affected individuals, and society as a whole. Opioid withdrawal is characterized by strong physical symptoms, along with signs of negative affect. Negative affect due to opioid withdrawal is a major obstacle to recovery and relapse prevention. The mechanisms behind negative affect due to either spontaneous or antagonist-precipitated opioid withdrawal are not well known, and more animal models need be developed. Here, we present behavioral models of negative affect upon naloxone-precipitated morphine withdrawal in adult male mice. Social, anxiety, and despair-like deficits were investigated following naloxone administration in mice receiving morphine under three dosing regimens; acute, chronic constant dose and chronic escalating doses. Social behaviour in the three-chamber social preference test was decreased following withdrawal from chronic and escalating but not acute morphine. Anxiety-like behaviour in the open field was increased for all three treatments. Despair-like behaviour was increased following withdrawal from chronic and escalating but not acute morphine. Altogether, these animal models will contribute to study behavioural and neuronal circuitries involved in the several negative affective signs characterizing OUD.

Opioid use disorder (OUD) is a complex relapsing disorder following repeated opioid exposure, affecting 16 million people worldwide with an estimation of 120,000 deaths worldwide annually attributed to OUD². OUD leads to significant impairment and poor life quality<fnref n="1" />,². OUD is often described within a recurring three-stage framework comprised of binge/intoxication upon drug consumption, withdrawal and negative mood when access to the drug is limited, and preoccupation/anticipation with the drug until the next intoxication³. Withdrawal is particularly problematic in OUD, as avoidance of the negative emotional state by using the drugs is a major negative reinforcer for continued opioid use⁴. Opioid withdrawal syndrome is comprised of an intense acute phase that develops within minutes to several hours after a cessation or reduction in opioid use⁵. Acute opioid withdrawal is characterized by dysphoria and somatic signs such as nausea or vomiting; muscle aches; lacrima-tion, pupillary dilation, piloerection, or sweating, diarrhea, yawning, fever, or insomnia⁵. Acute withdrawal can also be induced by administration of an opioid antagonist such as naloxone and naltrexone after chronic opioid use. Naloxone is a life-saving drug used to reverse opioid overdose⁶. However, a concern with the administration of naloxone in an opioid-dependent state is the precipitation of the highly aversive emotional and somatic with-drawal symptoms. The severity of signs of withdrawal is correlated to continued opioid use and risk of relapse⁷. It is therefore critical to alleviate the acute withdrawal phase and its associated negative affective state in people with OUDs, and it remains key to maintain abstinence.

The neurobiological mechanisms underlying negative affect during naloxone-precipitated withdrawal are still not well understood, however animal models have proven an invaluable preclinical tool to help advance the field⁸,⁹. Many animal studies of addiction-like behaviours aim to model the rewarding aspects of OUD and/or on the negative reinforcement, 'the dark side of addiction'<fnref n="1" />. Dysphoric mood during opioid withdrawal has been modelled in animals through avoidance of withdrawal-associated contexts, social deficits, and increased anxiety- and despair-like behaviours⁸. Most models of negative affect during opioid withdrawal are studied after chronic opioid exposure in order to induce the longer-term adaptations thought to be necessary for the

<footnote n="1">¹Université de Strasbourg (UNISTRA), INSERM UMR-S 1329, Strasbourg Translational Neuroscience and Psychiatry, Centre de Recherche en Biomédecine de Strasbourg, 1 rue Eugène Boeckel, 67084 Strasbourg Cedex, France. ²Department of Psychiatry, Douglas Hospital Research Center, McGill University, Montreal, Canada. ✉email: edarcq@unistra.fr</footnote>
development of a withdrawal signs⁸. However, few models address acute effects of morphine, though studies show that even a single administration of morphine can cause anxiety-like behaviours following naloxone precipitated withdrawal¹⁰,¹¹. Additionally, animal models typically include escalating dose opioid administration to replicate the increased opioid intake pattern in humans following the development of tolerance, however it is not often shown that escalating dosing is necessary for the development of negative affect. It is therefore important to distinguish negative affective signs that develop as a result of repeated and escalating opioid administration, in an effort to better model patterns of opioid intake.

Here, we present models of negative affect during naloxone-precipitated withdrawal from acute, chronic, and escalating dose morphine. Following naloxone-precipitated withdrawal from three different patterns of morphine exposure, mice were placed in behavioural tests of negative affect including social, anxiety-like and despair on three separate days. We showed that acute, chronic and escalating morphine were sufficient to induce increased anxiety-like behaviour. However, only chronic morphine, at constant and escalating dosing, induced social and despair-like behavioural deficits, indicating these behaviours are chronic dosing dependent features of negative affect. The negative affective deficits did not require escalating dose of morphine, and chronic opioid exposure was sufficient for all signs of negative affect tested. Somatic withdrawal symptoms in response to naloxone increased in severity as morphine dosing increased across the three administration patterns. Remarkably, while the presence of somatic withdrawal signs is typically used as a control for the development of physical dependence to morphine, they were also found to be increased following an acute dose of morphine. Thus, this comparison highlights the importance of these animal models for examining the neural and behavioural circuits and to test new therapeutic strategies related to negative emotional processes in the context of OUD.

# Materials and methods

## Mice

Adult male C57Bl6/J mice (> 8 weeks old; Charles River) were housed in groups of 4–5 in a temperature and humidity-controlled room. Juvenile C57Bl/6J mice (< 7 weeks old; Charles River) were used as interactors for test mice in the social preference test. All experiments were performed in accordance with the ARRIVE guidelines. All procedures in this report were conducted in accordance with the relevant guidelines and regulation set forth by the Canadian Council of Animal Care and by the Animal Care Committees of McGill University/Douglas Mental Health University Institute and were also approved by the Regional Committee of Ethic in Animals Experiment of Strasbourg (CREMEAS).

## Drugs

All the drugs were injected intraperitoneally (i.p.). In the acute morphine study (Fig. 1A), all mice received a saline injection (0.9%) once daily for 5 days. On day 6, mice received an acute injection of morphine (20 mg/kg) or saline (equivalent in volume), and 30 min later all mice received an injection of naloxone (2 mg/kg). In the chronic morphine study (Fig. 1B), mice received morphine (20 mg/kg) or equivalent in volume of saline once daily for 5 days. On day 6, mice received an injection of morphine (20 mg/kg) or saline, and 30 min later all mice received an injection of naloxone (2 mg/kg). In the escalating chronic morphine study (Fig. 1C), mice received morphine at escalating doses (day 1, 10 mg/kg; day 2, 20 mg/kg; day 3, 30 mg/kg; day 4, 40 mg/kg; day 5, 50 mg/kg) or equivalent in volume of saline once daily for 5 days. On day 6, mice received an injection of morphine (50 mg/kg) or saline, and 30 min later all mice received an injection of naloxone (2 mg/kg). In all experiments, drug administrations of day 6 were repeated on days 7 and 8.

## Behaviour of negative affect

In all studies, mice were placed in behavioural tests 30 min after naloxone injection on experimental days 6, 7 and 8. In all studies; on day 6, mice were placed in a social preference test (SPT)¹²,¹³, on day 7 an open field test (OFT)¹⁴, and on day 8 a tail suspension test (TST)¹⁵. Additionally, on day 6, mice were scored for somatic withdrawal signs for 15-min after naloxone. Detailed descriptions of behavioural procedures are in the Supplementary Information and as described¹²⁻¹⁵. This includes the description of precipitated withdrawal sign scoring, SPT, OFT, and TST.

## Statistics

Data are presented as mean ± SEM. Global withdrawal sign scoring and immobility time in the TST were represented across time and were analyzed using RM two-way ANOVAs. Significant main effects (p ≤ 0.05) were followed by Bonferroni's multiple comparisons tests. All other experiments were first tested for normality with a Shapiro-Wilk test, followed by an unpaired t-test or Mann-Whitney U test. P-values < 0.05 were considered statistically significant. All data was analyzed using GraphPad Prism (v9) software. Please refer to Supplementary Information Table 1 for a description of all statistical tests.

# Results

## Three modes of morphine administration to study negative affect of withdrawal

Here, we designed models of negative affect during naloxone-precipitated withdrawal from acute (Fig. 1A), chronic, constant (Fig. 1B) and escalating (Fig. 1C) doses morphine. Following naloxone-precipitated withdrawal from three different patterns of morphine exposure, negative affect was evaluated using social, anxiety-like and despair behavioural tests on three separate days.
<figure><img src="image_3.png" /><figcaption>**Figure 1.** Experimental timeline of models of negative affect following naloxone-precipitated withdrawal from acute or chronic morphine. (A) Scheme of experimental timeline for naloxone-precipitated withdrawal from acute morphine. (B,C) Scheme of experimental timeline for naloxone-precipitated withdrawal from chronic morphine [with constant (B) or escalating (C) doses].</figcaption></figure>

## Both acute and chronic morphine treatments induce signs of somatic withdrawal in naloxone administered mice

Somatic withdrawal signs are one of the most widely reported signs of naloxone-precipitated opioid withdrawal in humans, and have also been well described in rodent models⁸. In order to evaluate how different morphine regimens induce somatic withdrawal signs following naloxone injection, we carried out withdrawal sign scoring following acute (Fig. 1A) or 5-day chronic morphine dosing (Fig. 1B). Mice were injected with naloxone 30-min after morphine and immediately placed in a tall arena for 15-min while they were scored by observers blind to treatment group. Withdrawal from acute and chronic morphine induced a higher withdrawal sign score (see Supplementary Information methods for formula) compared to saline controls (Fig. 2C, *p = 0.01; Fig. 2E, t₂₇ = 5.487, ****p < 0.0001). When withdrawal scores were analyzed over 5-min time bins, only chronic morphine mice displayed significantly increased withdrawal signs in the first 5-min, and both acute and chronic morphine treated mice differed significantly from saline controls from 5 to 10 min (Fig. 2D, main effect of treatment, F₁,₁₉ = 8.63, **p < 0.01; Fig. 2F, main effect of treatment, F₁,₂₇ = 31.13, ****p < 0.0001). At the end of the 15-min of withdrawal scoring, neither acute nor chronic morphine treated mice differed from their respective saline controls (Fig. 2D,F). Both acute and chronic precipitated morphine withdrawal induced typical signs of withdrawal including decreased horizontal activity (Fig. 2G, p = 0.0504; Fig. 2I, **p < 0.01), increased the number of jumping (Fig. 2H, *p < 0.05; Fig. 2J, ****p < 0.0001) and paw tremor events (Fig. 2K, *p < 0.05; Fig. 2M, *p < 0.05). Withdrawal from acute morphine induced no changes in ptosis, teeth chattering, piloerection or sniffing (Fig. 2L, p = 0.86; Fig. 2O, p = 0.48; Fig. 2P, p = 0.09; Fig. 2S, p = 0.48). Withdrawal from chronic morphine induced increased ptosis, teeth chattering, piloerection and sniffing events (Fig. 2N, ****p < 0.001; Fig. 2Q, ****p < 0.001; Fig. 2R, ****p < 0.001; Fig. 2U, *p < 0.05). Additionally, withdrawal from acute and not chronic increased the number of head shakes (Fig. 2T, *p < 0.05; Fig. 2V, *p = 0.1). Finally, both acute and chronic morphine decreased rearing events and grooming events (Supplementary Information Fig. 1), the latter an indicator of decreased self-directed behaviour, often present in mouse models of negative affect such as the unpredictable chronic mild stress model¹⁶. These results show that both acute and chronic morphine induce increased signs of somatic withdrawal after naloxone.

## Chronic but not acute morphine treatment induces social deficits during naloxone precipitated withdrawal in mice

Social isolation during opioid withdrawal is a major impediment to recovery and relapse prevention⁹. We tested whether naloxone-precipitated withdrawal from acute or chronic morphine in mice could induce social deficits
<figure><img src="image_4.png" /><figcaption>C. Global Withdrawal</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>D. Global Withdrawal</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>G. Horizontal Activity</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>H. Jumping</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>K. Paw Tremors</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>L. Ptosis</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>O. Teeth Chattering</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>P. Piloerection</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>S. Sniffing</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>T. Head Shakes</figcaption></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /><figcaption>E. Global Withdrawal</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>F. Global Withdrawal</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>I. Horizontal Activity</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>J. Jumping</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>M. Paw Tremors</figcaption></figure>

<figure><img src="image_20.png" /><figcaption>N. Ptosis</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>Q. Teeth Chattering</figcaption></figure>

<figure><img src="image_22.png" /><figcaption>R. Piloerection</figcaption></figure>

<figure><img src="image_23.png" /><figcaption>U. Sniffing</figcaption></figure>

<figure><img src="image_24.png" /><figcaption>V. Head Shakes</figcaption></figure>

<figure><img src="image_25.png" /></figure>

**Figure 2.** Naloxone-precipitated somatic withdrawal signs in acute and chronic morphine-administered mice (A,B) Schematic representation of experimental timelines of naloxone-precipitated withdrawal sign scoring for acute and chronic morphine-treated mice, respectively. (C,E) Increased global withdrawal sign score after naloxone injection in acute and chronic morphine treated mice, respectively. (D,F) Withdrawal sign scoring in 5-min time bins after naloxone injection in acute (D) and chronic (F) morphine treated mice. (G,I) Decreased horizontal activity score after naloxone injection in acute and chronic morphine treated mice, respectively. (H,J) Increased number of jumping events after naloxone injection in acute and chronic morphine treated mice, respectively. (K,M) Increased number of paw tremors after naloxone injection in acute and chronic morphine treated mice, respectively. (L,N) Ptosis score after naloxone injection is unaltered in acute (L) and increased in chronic (N) morphine treated mice. (O,Q) Number of teeth chattering events after naloxone injection is unaltered in acute (O) and increased in chronic (Q) morphine treated mice. (P,R) Piloerection score is not affected after naloxone injection in acute (P) and is increased in chronic morphine (R) treated mice. (S,U) Number of sniffing events is not affected after naloxone injection in acute (S) and is increased in chronic morphine (U) treated mice. (T,V) Number of head shakes is increased after naloxone injection in acute (T) but not in chronic morphine (V) treated mice. Data are represented as mean ± SEM. Dots represent individual values. \*p<0.05, \*\*p<0.01, \*\*\*p<0.001, \*\*\*\*p<0.0001. N=10-15/group.
mice treated prior with acute or chronic morphine were placed in an open field arena 30-min following naloxone administration (Fig. 4A,B). For 15 min, the time spent in the center of the arena, the number of entries to the center zone and the total distance travelled were recorded. Acute morphine treated mice spent less time in the center compared to saline controls during naloxone-precipitated withdrawal (Fig. 4C; t₁₉ = 3.25, **p < 0.01), demonstrating increased anxiety-like behaviour with no effect on locomotion (Fig. 4E; p = 0.70). There was no effect of withdrawal from acute morphine on the number of entries to the center of the arena (Fig. 4D; t₁₉ = 1.73, p = 0.0990). Chronic morphine treated mice also display increased anxiety-like behaviour during naloxone-precipitated withdrawal, demonstrated by decreased time in the center (Fig. 4F; t₂₅ = 3.65, **p < 0.01) and number entries to the center (Fig. 4G; *p < 0.05). However, precipitated withdrawal from chronic morphine also decreased locomotor activity demonstrated by lower distance travelled (Fig. 4H; t₂₅ = 2.48, *p < 0.05). Our results suggest that precipitated-withdrawal from acute and chronic morphine increase anxiety-like behaviour in the OFT, modeling a commonly reported symptom present during naloxone-precipitated withdrawal in humans.

## Chronic but not acute morphine treatment induces despair-like behaviour during naloxone precipitated withdrawal in mice

To further probe the effect of morphine withdrawal on the spectrum of negative affect, we investigated the effect of naloxone-precipitated withdrawal from acute and chronic morphine on despair-like behaviour measured by the TST. Thirty minutes after a naloxone injection to precipitate withdrawal from acute or chronic morphine, mice were suspended upside-down from a hook by a paper clip attached to the tip of their tails (Fig. 5A,B). Total immobility time over 6 min was measured automatically by the apparatus. Mice withdrawn from acute morphine showed no difference in immobility time from saline controls (Fig. 5C, t₁₉ = 0.50, p = 0.62; Fig. 5D, main effect of treatment, F(1, 19) = 0.25, p = 0.62). However, we found that precipitated withdrawal from chronic morphine increased immobility time, showing deficits in active coping in the tail suspension test (Fig. 5E, t₂₃ = 3.32, **p < 0.01; Fig. 5F, main effect of treatment, F(1, 23) = 11.01, **p < 0.01). These results suggest that precipitated-withdrawal from chronic but not acute morphine is necessary to induce despair-like behaviour measured by the TST.

## Naloxone-precipitated withdrawal from escalating chronic morphine induces somatic withdrawal signs

One of the major characteristics of opioid addiction is a development of tolerance over time, leading to an escalation of opioid dose used¹. To model this pattern of opioid intake, we used a mouse model of an escalating 5-day chronic treatment of morphine in order to evaluate the severity of somatic withdrawal signs following naloxone injection (Fig. 6A). Mice were injected with naloxone 30-min after morphine and somatic withdrawal signs were scored. Withdrawal from chronic escalating morphine induced a high global withdrawal sign score compared to saline controls (Fig. 6B, U = 0, ****p < 0.0001). When withdrawal scores were analyzed over 5-min time bins, chronic escalating morphine treated mice displayed significantly increased withdrawal signs compared to saline controls over all 5-min time bins (Fig. 6C, main effect of treatment, F(1, 21) = 131.1, ****p < 0.0001). At the end of the 30 min of withdrawal scoring, escalating morphine treated mice did not differ from saline controls (Supplementary Information Fig. 2). Precipitated withdrawal from chronic escalating morphine induced typical signs of withdrawal including decreased horizontal activity (Fig. 6D, U = 0, ****p < 0.0001), increased the number of jumping (Fig. 6E, U = 15, ****p < 0.001), paw tremor (Fig. 6F, U = 13.5, ****p < 0.001) and teeth chattering events (Fig. 6H, U = 5.5, ****p < 0.0001). Withdrawal from chronic escalating morphine induced increased ptosis and

<figure><img src="image_26.png" /></figure>

**Figure 4.** Naloxone-induced anxiety-like behaviour in acute and chronic morphine-administered mice. (A,B) Schematic representation of experimental timelines of open field test for acute (i.p.) and chronic (i.p.) morphine-treated mice, respectively. (C,F) Decreased time in centre zone in the open field test during naloxone-precipitated withdrawal from acute and chronic morphine, respectively. (D,E) No effect of precipitated withdrawal from acute morphine on entries to centre and distance travelled in the open field test, respectively. (G,H) Decreased entries to centre zone and distance travelled in the open field test during naloxone-precipitated withdrawal from chronic morphine. Data are represented as mean ± SEM. Dots represent individual values.
*p < 0.05, **p < 0.01. N = 10–15/group.
<figure><img src="image_27.png" /><figcaption>**Figure 5.** Naloxone-induced despair-like behaviour in chronic but not acute morphine-administered mice (A,B) Schematic representation of experimental timelines of tail suspension test for acute (i.p.) and chronic (i.p.) morphine-treated mice, respectively. (C,D) No effect naloxone-precipitated withdrawal from acute morphine on immobility time in the tail suspension test. (C,D) Increased immobility time in the tail suspension test during naloxone-precipitated withdrawal from chronic morphine. Data are represented as mean ± SEM. Dots represent individual values. \*p<0.05, \*\*p<0.01. N = 10–15/group.</figcaption></figure>

piloerection scores (Fig. 6G, U = 0, \*\*\*\*p < 0.0001; Fig. 6I, U = 0, \*\*\*\*p < 0.0001). Additionally, withdrawal from chronic escalating morphine had no significant effect on the number of sniffing and head shake events (Fig. 6J, U = 44, p = 0.09; Fig. 6K, U = 66, p > 0.99). Finally, escalating morphine decreased rearing and grooming events (Supplementary Information Fig. 1). These results show that as with acute and chronic morphine, escalating morphine induces increased signs of somatic withdrawal after naloxone. Escalating morphine treated mice displayed higher and more long-lasting global withdrawal signs when compared to acute and chronic constant morphine treated mice. Additionally, escalating treatment increased jumping, paw tremor, teeth chattering and ptosis scores when compared to acute and constant morphine. While acute morphine increased head shakes after naloxone, and constant morphine increased sniffing, escalating morphine had no significant effect on these signs, demonstrating a dosing paradigm-dependent apparition of withdrawal signs.

## Naloxone-precipitated withdrawal from escalating chronic morphine induces social deficits, anxiety-like and despair-like behaviour

Next, we determine whether the negative affect deficits are also present after an escalating opioid treatment. To do so we used a mouse model of an escalating 5-day chronic treatment of morphine and tested negative affect during naloxone precipitated withdrawal (Fig. 7A). After a 5-day escalating morphine treatment, mice were injected with naloxone and subjected to a series of behavioural tests probing negative affect over the course of 3 days. Social behaviour was tested on day 6 with an SPT 30 min after naloxone injection. Both saline and morphine-treated mice showed no preference for either chamber during the habituation phase (Fig. 7B; t$_{21}$ = 0.03, p = 0.98). Precipitated withdrawal from escalating morphine decreased time spent in the social chamber when compared to saline controls (Fig. 7C; t$_{21}$ = 2.48, \*p < 0.05). On day 7, mice were then placed in an OFT 30-min after naloxone to test anxiety-like behaviour. Escalating chronic morphine treated mice displayed increased anxiety-like behaviour during naloxone-precipitated withdrawal, demonstrated by decreased time in the center (Fig. 7D; t$_{22}$ = 2.63, \*p < 0.05) and number entries to the center of the arena (Fig. 7E; t$_{22}$ = 2.30, \*p < 0.05); with no effect on locomotion (Fig. 7F; t$_{21}$ = 0.11, p = 0.92). Finally, on day 8 mice were placed in a TST to measure despair-like behaviour 30-min after naloxone. Precipitated withdrawal from escalating chronic morphine increased immobility time, demonstrating deficits in active coping in the tail suspension test (Fig. 7G, t$_{22}$ = 2.83, \*\*p < 0.01; Fig. 7H, main effect of treatment, F$_{1,22}$ = 7.99, \*\*p < 0.01). These results suggest that this model of naloxone precipitated withdrawal from chronic escalating morphine, as after chronic constant morphine, is able to model social deficits and anxiety-like and despair-like behavioural signs of negative affect.

## Discussion

Here, we established and described a model of adverse effects during and after naloxone-induced withdrawal from morphine with acute, chronic, and increasing regiments. Mice were subjected to behavioural measures of negative affect, which included social interaction, anxiety-like, and despairing tasks. We demonstrated that enhanced anxiety-like behaviour could be induced by both acute and chronic morphine use. Nevertheless, social and depressive-like behavioural abnormalities were only brought on by chronic dosing, suggesting that these behaviours are chronic dosing dependent aspects of negative affect. Chronic opioid exposure was sufficient to produce all tested indicators of negative affect, and increasing the amount of morphine did not increase the negative affective impairments.
<figure><img src="image_28.png" /></figure>

<figure><img src="image_29.png" /></figure>

<figure><img src="image_30.png" /></figure>

<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /></figure>

<figure><img src="image_37.png" /></figure>

<figure><img src="image_38.png" /><figcaption>**Figure 6.** Naloxone-precipitated somatic withdrawal signs in chronic escalating morphine-administered mice (A) Schematic representation of experimental timeline of naloxone-precipitated withdrawal sign scoring for chronic escalating morphine-treated mice. (B) Increased global withdrawal sign score after naloxone injection in escalating morphine treated mice. (C) Withdrawal sign scoring in 5-min time bins after naloxone injection in escalating morphine treated mice. (D) Decreased horizontal activity score after naloxone injection in escalating morphine treated mice. Increased number of jumping (E), paw tremors (F), teeth chattering events (H) after naloxone injection in escalating morphine treated mice. Ptosis (G) and piloerection (I) scores are increased after naloxone injection in escalating morphine treated mice. Number of sniffing (J) and head shake (K) events is not affected after naloxone injection in chronic escalating morphine treated mice. Data are represented as mean ± SEM. Dots represent individual values. ***p<0.001, ****p<0.0001. N=11-13/group.</figcaption></figure>
A. Escalating morphine

<figure><img src="image_39.png" /></figure>

<figure><img src="image_40.png" /></figure>

<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /></figure>

<figure><img src="image_43.png" /></figure>

<figure><img src="image_44.png" /></figure>

<figure><img src="image_45.png" /></figure>

<figure><img src="image_46.png" /></figure>

**Figure 7.** Negative affect during naloxone-precipitated withdrawal in mice receiving a chronic escalating dose morphine regimen. (A) Scheme of experimental procedure. (B,C) Decreased social preference during naloxone-precipitated withdrawal from escalating morphine. (D–F) Decreased time and entries to the centre zone in the open field following naloxone-precipitated withdrawal from escalating morphine with no effect on distance travelled. (H,I) Increased immobility time in the tail suspension test during naloxone-precipitated withdrawal from chronic escalating morphine. \*p<0.05, \*\*p<0.01. Data are represented as mean ± SEM. Dots represent individual values. N = 11–13/group.
## Other animal models of antagonist-precipitated withdrawal

When an opioid-dependent individual receives an opioid receptor antagonist, it causes a withdrawal episode¹⁷. Naltrexone and naltrexone are the two primary opioid antagonists that are commonly utilized in the clinical context. One of the three FDA-approved drugs for treating OUDs is naltrexone, which is also used to reduce opioid overdose deaths¹⁸. Exposure to antagonists causes and intensifies the withdrawal symptoms that naturally happen when the opioid medicine wears off. Withdrawal symptoms (somatic and negative affect) can be enhanced and temporally controlled using antagonist precipitated opioid withdrawal models. Inducing somatic withdrawal symptoms in dogs and rodents through antagonist-precipitated withdrawal has been a popular method for confirming the existence of physical dependency¹⁹⁻²³. After just one opioid encounter, agonist-precipitated withdrawal symptoms can be quantified²⁴. This phenomena was found in dogs exhibiting antagonist-precipitated somatic withdrawal-like symptoms one hour following a single morphine administration¹⁹ and in human and rodents²⁴⁻²⁶. The amount of withdrawal symptoms following an acute dosage is largely influenced by the agonist and antagonist doses as well as the time between the two administrations¹,²⁴. When an antagonist is administered following an acute morphine dose, anxiety-like reactions are enhanced for up to 80 days following the acute withdrawal phase (two hours)¹⁰,¹¹. And as here, several symptoms of antagonist-precipitated withdrawal, such as aversion to withdrawal-conditioned taste and location avoidance¹⁰,²⁷⁻²⁹, increased anxiety-like responses¹⁰,¹¹,²⁷,³⁰⁻³⁴, and anhedonia and reward deficits³⁵⁻³⁷, that were observed under chronic opioid treatments were also present following a single, unusual acute dosage. Altogether, these and our studies suggest that negative affect after withdrawal from an acute opioid dose may be similar to signs of withdrawal from chronic opioid.

## Somatic precipitated withdrawal signs are present after both acute and chronic morphine

Typically, the presence of somatic withdrawal signs has been used in animal models of addiction to verify the presence of physical dependence³⁸. Our study demonstrates that one acute dose of morphine 30 min prior to naloxone is sufficient to induce somatic withdrawal signs, therefore the use of naloxone as an indicator of dependence that develops as a result of chronic exposure to morphine might not be always necessary. However, chronic morphine withdrawal induced higher severity of signs of withdrawal, likely reflecting the adaptations that occur at the cellular level over a chronic treatment. Finally, chronic escalating morphine withdrawal induced the most severe and long-lasting somatic withdrawal signs, demonstrating a dose-dependent increase in severity and duration of withdrawal signs. Importantly, neither acute nor chronic constant morphine treated mice differed from their saline controls by the end of the 15-min scoring period, while chronic escalating morphine treated mice did not differ from controls at the 25-min time point (Supplementary Information Fig. 2). This demonstrates that somatic withdrawal signs were absent 30 min after naloxone, preventing interference with behavioural tests of negative affect.

## Social and despair-deficits

Social deficits are a hampering symptom of opioid withdrawal. The literature focused on social behavioural deficits following protracted abstinence in mice³⁸,³⁹, with few studies on social behaviour following short-term opioid withdrawal¹². In one study, thirty minutes after naloxone injection, time in the social zone during the social preference test was reduced by naloxone-precipitated morphine withdrawal¹². While our results are coherent with this previous study, social interaction deficits may be more easily and robustly detected using live mouse tracker technology allowing a detection of social interaction on an automatic and unbiased manner⁴⁰. Importantly, only precipitated withdrawal from chronic morphine could induce social and despair-like deficits. These signs are appropriate to test for adaptations after long-term opioid use and mechanisms behind withdrawal.

## Anxiety-like behaviour deficits

Antagonist-precipitated opioid withdrawal from chronic opioids induces anxiety-like behavioural deficits. The unpleasant withdrawal experience was often associated to a cue or context²³,⁴¹,⁴². Anxiety-like deficits were also found after antagonist-precipitated withdrawal in the elevated plus maze³⁴,⁴³. Furthermore, this anxiety-like behavioural deficits are long lasting, as Rothwell and colleagues showed that naloxone-precipitated withdrawal increased anxiety-like responses until 80 days after the exposure to one morphine (10 mg/kg) injection¹⁰. Anxiety-like behaviour is a commonly detected sign of negative affect in many rodent models of withdrawal⁸. Here, anxiety is detected following withdrawal from each morphine treatment, demonstrating a sensitive sign of negative affect present after withdrawal from even acute morphine. In the literature, anxiety is present after short-term withdrawal and protracted abstinence from acute and chronic opioid exposure⁸. Withdrawal from an acute morphine dose as presented here could serve as an appropriate model to detect early adaptations to opioids that are revealed by mu-opioid receptor antagonism.

## Improvement and use of the model

To more accurately mimic patterns of opioid intake, it is crucial to differentiate between unpleasant emotional signals that arise from recurrent and increasing opioid administration. It may be important to develop a model of antagonist precipitated withdrawal after voluntary intake of opioid to better mimic the pattern of opioid consumption in humans. Other studies, show that there was no discernible impact on heroin-seeking behaviour in dependent or non-dependent rats when opioid antagonists (naltrexone or naloxone) created an unpleasant condition⁴⁴⁻⁴⁶. Naloxone-precipitated somatic signs of withdrawal were detected in rats that self-administered heroin⁴⁷. Naloxone also increases voluntary heroin intake in operant self-administration procedure⁴⁸,⁴⁹. It is also important to note that the number of naloxone administrations may impact the results, and thus the intensity of negative affect deficits may depend of the order of the behavioral tests. Another limitation of the study is that sex differences were not studied as only male mice were used. Indeed, sex may influence negative affective deficits. It
was shown previously, that sex influences some signs of despair or anxiety after protracted withdrawal⁵⁰. Another study shows no main difference in withdrawal signs between males and females after naloxone-precipitated withdrawal from acute morphine (besides number of fecal boli, paw tremor and weight loss)⁷. Thus, further studies will be necessary to determine the impact of the sex on negative affect after opioid withdrawal.

Altogether, the model describes in our study will be important to dissect the neural and behavioural circuits and to test new therapeutic strategy involved in the development of negative affect in the context of OUD.

## Data availability

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Received: 29 December 2023; Accepted: 25 April 2024

Published online: 29 April 2024

## References

1. Strang, J. et al. Opioid use disorder. *Nat. Rev. Dis. Primers* **6**, 3. https://doi.org/10.1038/s41572-019-0137-5 (2020).

2. Dydyk, A. M., Jain, N. K. & Gupta, M. In *StatPearls* (2023).

3. Koob, G. F. & Volkow, N. D. Neurobiology of addiction: A neurocircuitry analysis. *Lancet Psychiatry* **3**, 760–773. https://doi.org/10.1016/S2215-0366(16)00104-8 (2016).

4. Koob, G. F. Neurobiology of opioid addiction: Opponent process, hyperkatifeia, and negative reinforcement. *Biol. Psychiatry* **87**, 44–53. https://doi.org/10.1016/j.biopsych.2019.05.023 (2020).

5. American Psychiatric, A. *Diagnostic and Statistical Manual of Mental Disorders: DSM-5*. [DSM V], 5th edn (American Psychiatric Publishing, 2013).

6. Rzasa Lynn, R. & Galinkin, J. L. Naloxone dosage for opioid reversal: Current evidence and clinical implications. *Ther. Adv. Drug Saf.* **9**, 63–88. https://doi.org/10.1177/2042098617744161 (2018).

7. Luster, B. R. et al. Inhibitory transmission in the bed nucleus of the stria terminalis in male and female mice following morphine withdrawal. *Addict. Biol.* **25**, e12748. https://doi.org/10.1111/adb.12748 (2020).

8. Ozdemir, D., Allain, F., Kieffer, B. L. & Darcq, E. Advances in the characterization of negative affect caused by acute and protracted opioid withdrawal using animal models. *Neuropharmacology* **232**, 109524. https://doi.org/10.1016/j.neuropharm.2023.109524 (2023).

9. Welsch, L., Bailly, J., Darcq, E. & Kieffer, B. L. The negative affect of protracted opioid abstinence: Progress and perspectives from rodent models. *Biol. Psychiatry* **87**, 54–63. https://doi.org/10.1016/j.biopsych.2019.07.027 (2020).

10. Rothwell, P. E., Thomas, M. J. & Gewirtz, J. C. Protracted manifestations of acute dependence after a single morphine exposure. *Psychopharmacology (Berl)* **219**, 991–998. https://doi.org/10.1007/s00213-011-2425-y (2012).

11. Zhang, Z. & Schulteis, G. Withdrawal from acute morphine dependence is accompanied by increased anxiety-like behavior in the elevated plus maze. *Pharmacol. Biochem. Behav.* **89**, 392–403. https://doi.org/10.1016/j.pbb.2008.01.013 (2008).

12. Valentinova, K. et al. Morphine withdrawal recruits lateral habenula cytokine signaling to reduce synaptic excitation and sociability. *Nat. Neurosci.* **22**, 1053–1056. https://doi.org/10.1038/s41593-019-0421-4 (2019).

13. Allain, F., Carter, M., Dumas, S., Darcq, E. & Kieffer, B. L. The mu opioid receptor and the orphan receptor GPR151 contribute to social reward in the habenula. *Sci. Rep.* **12**, 20234. https://doi.org/10.1038/s41598-022-24395-z (2022).

14. Bailly, J. et al. Habenular neurons expressing mu opioid receptors promote negative affect in a projection-specific manner. *Biol. Psychiatry* **93**, 1108–1117. https://doi.org/10.1016/j.biopsych.2022.09.013 (2023).

15. Welsch, L. et al. Mu opioid receptor-expressing neurons in the dorsal raphe nucleus are involved in reward processing and affective behaviors. *Biol. Psychiatry* https://doi.org/10.1016/j.biopsych.2023.05.019 (2023).

16. Nollet, M. Models of depression: Unpredictable chronic mild stress in mice. *Curr. Protoc.* **1**, e208. https://doi.org/10.1002/cpz1.208 (2021).

17. Kanof, P. D. et al. Clinical characteristics of naloxone-precipitated withdrawal in human opioid-dependent subjects. *J. Pharmacol. Exp. Ther.* **260**, 355–363 (1992).

18. Volkow, N. D. & Blanco, C. Medications for opioid use disorders: Clinical and pharmacological considerations. *J. Clin. Investig.* **130**, 10–13. https://doi.org/10.1172/jci134708 (2020).

19. Wikler, A. & Carter, R. L. Effects of single doses of N-Allylnormorphine on hindlimb reflexes of chronic spinal dogs during cycles of morphine addiction. *J. Pharmacol. Exp. Ther.* **109**, 92–101 (1953).

20. Martin, W. R., Eades, C. G., Thompson, W. O., Thompson, J. A. & Flanary, H. G. Morphine physical dependence in the dog. *J. Pharmacol. Exp. Ther.* **189**, 759–771 (1974).

21. Blasig, J. & Herz, A. Precipitated morphine withdrawal in rats as a tool in opiate research. *Curr. Dev. Psychopharmacol.* **4**, 129–149 (1977).

22. Matthes, H. W. et al. Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the mu-opioid-receptor gene. *Nature* **383**, 819–823. https://doi.org/10.1038/383819a0 (1996).

23. Frenois, F., Cador, M., Caille, S., Stinus, L. & Le Moine, C. Neural correlates of the motivational and somatic components of naloxone-precipitated morphine withdrawal. *Eur. J. Neurosci.* **16**, 1377–1389. https://doi.org/10.1046/j.1460-9568.2002.02187.x (2002).

24. Harris, A. C. & Gewirtz, J. C. Acute opioid dependence: Characterizing the early adaptations underlying drug withdrawal. *Psychopharmacology (Berl)* **178**, 353–366. https://doi.org/10.1007/s00213-005-2155-0 (2005).

25. Meyer, D. R. & Sparber, S. B. Evidence of possible opiate dependence during the behavioral depressant action of a single dose of morphine. *Life Sci.* **21**, 1087–1093. https://doi.org/10.1016/0024-3205(77)90106-0 (1977).

26. Bickel, W. K., Stitzer, M. L., Liebson, I. A. & Bigelow, G. E. Acute physical dependence in man: Effects of naloxone after brief morphine exposure. *J. Pharmacol. Exp. Ther.* **244**, 126–132 (1988).

27. Radke, A. K. & Gewirtz, J. C. Increased dopamine receptor activity in the nucleus accumbens shell ameliorates anxiety during drug withdrawal. *Neuropsychopharmacology* **37**, 2405–2415. https://doi.org/10.1038/npp.2012.97 (2012).

28. Parker, L. A., Cyr, J. A., Santi, A. N. & Burton, P. D. The aversive properties of acute morphine dependence persist 48 h after a single exposure to morphine: Evaluation by taste and place conditioning. *Pharmacol. Biochem. Behav.* **72**, 87–92. https://doi.org/10.1016/S0091-3057(01)00724-9 (2002).

29. Azar, M. R., Jones, B. C. & Schulteis, G. Conditioned place aversion is a highly sensitive index of acute opioid dependence and withdrawal. *Psychopharmacology (Berl.)* **170**, 42–50. https://doi.org/10.1007/s00213-003-1514-y (2003).

30. Harris, A. C. & Gewirtz, J. C. Elevated startle during withdrawal from acute morphine: A model of opiate withdrawal and anxiety. *Psychopharmacology (Berl.)* **171**, 140–147. https://doi.org/10.1007/s00213-003-1573-0 (2004).
31. Harris, A. C., Atkinson, D. M., Aase, D. M. & Gewirtz, J. C. Double dissociation in the neural substrates of acute opiate dependence as measured by withdrawal-potentiated startle. *Neuroscience* **139**, 1201–1210. https://doi.org/10.1016/j.neuroscience.2006.01.048 (2006).

32. Cabral, A., Ruggiero, R. N., Nobre, M. J., Brandão, M. L. & Castilho, V. M. GABA and opioid mechanisms of the central amygdala underlie the withdrawal-potentiated startle from acute morphine. *Prog. Neuropsychopharmacol. Biol. Psychiatry* **33**, 334–344. https://doi.org/10.1016/j.pnpbp.2008.12.012 (2009).

33. Hamilton, K. L., Harris, A. C. & Gewirtz, J. C. Affective and neuroendocrine effects of withdrawal from chronic, long-acting opiate administration. *Brain Res.* **1538**, 73–82. https://doi.org/10.1016/j.brainres.2013.09.026 (2013).

34. McElligott, Z. A. et al. Noradrenergic synaptic function in the bed nucleus of the stria terminalis varies in animal models of anxiety and addiction. *Neuropsychopharmacology* **38**, 1665–1673. https://doi.org/10.1038/npp.2013.63 (2013).

35. Schulteis, G., Morse, A. C. & Liu, J. Repeated experience with naloxone facilitates acute morphine withdrawal: Potential role for conditioning processes in acute opioid dependence. *Pharmacol. Biochem. Behav.* **76**, 493–503. https://doi.org/10.1016/j.pbb.2003.09.006 (2003).

36. Schulteis, G., Heyser, C. J. & Koob, G. F. Opiate withdrawal signs precipitated by naloxone following a single exposure to morphine: Potentiation with a second morphine exposure. *Psychopharmacology* (Berl.) **129**, 56–65. https://doi.org/10.1007/s002130050162 (1997).

37. Amitai, N., Liu, J. & Schulteis, G. Discrete cues paired with naloxone-precipitated withdrawal from acute morphine dependence elicit conditioned withdrawal responses. *Behav. Pharmacol.* **17**, 213–222. https://doi.org/10.1097/00008877-200605000-00002 (2006).

38. Goeldner, C. et al. Impaired emotional-like behavior and serotonergic function during protracted abstinence from chronic morphine. *Biol. Psychiatry* **69**, 236–244. https://doi.org/10.1016/j.biopsych.2010.08.021 (2011).

39. Lalanne, L. et al. Kappa opioid receptor antagonism and chronic antidepressant treatment have beneficial activities on social interactions and grooming deficits during heroin abstinence. *Addict. Biol.* **22**, 1010–1021. https://doi.org/10.1111/adb.12392 (2017).

40. de Chaumont, F. et al. Real-time analysis of the behaviour of groups of mice via a depth-sensing camera and machine learning. *Nat. Biomed. Eng.* **3**, 930–942. https://doi.org/10.1038/s41551-019-0396-1 (2019).

41. Noe, E. et al. Arc reactivity in accumbens nucleus, amygdala and hippocampus differentiates cue over context responses during reactivation of opiate withdrawal memory. *Neurobiol. Learn. Mem.* **159**, 24–35. https://doi.org/10.1016/j.nlm.2019.02.007 (2019).

42. Boulos, L. J. et al. Mu opioid receptors in the medial habenula contribute to naloxone aversion. *Neuropsychopharmacology* **45**, 247–255. https://doi.org/10.1038/s41386-019-0395-7 (2020).

43. Schulteis, G., Yacke, M., Risbrough, V. & Koob, G. F. Anxiogenic-like effects of spontaneous and naloxone-precipitated opiate withdrawal in the elevated plus-maze. *Pharmacol. Biochem. Behav.* **60**, 727–731. https://doi.org/10.1016/s0091-3057(98)00034-3 (1998).

44. Stewart, J. & Wise, R. A. Reinstatement of heroin self-administration habits: Morphine prompts and naltrexone discourages renewed responding after extinction. *Psychopharmacology* (Berl.) **108**, 79–84. https://doi.org/10.1007/BF02245289 (1992).

45. Shaham, Y. & Stewart, J. Stress reinstates heroin-seeking in drug-free animals: An effect mimicking heroin, not withdrawal. *Psychopharmacology* (Berl.) **119**, 334–341. https://doi.org/10.1007/BF02246300 (1995).

46. Shaham, Y., Rajabi, H. & Stewart, J. Relapse to heroin-seeking in rats under opioid maintenance: The effects of stress, heroin priming, and withdrawal. *J. Neurosci.* **16**, 1957–1963. https://doi.org/10.1523/JNEUROSCI.16-05-01957.1996 (1996).

47. Vendruscolo, L. F. et al. Escalation patterns of varying periods of heroin access. *Pharmacol. Biochem. Behav.* **98**, 570–574. https://doi.org/10.1016/j.pbb.2011.03.004 (2011).

48. Kenny, P. J., Chen, S. A., Kitamura, O., Markou, A. & Koob, G. F. Conditioned withdrawal drives heroin consumption and decreases reward sensitivity. *J. Neurosci.* **26**, 5894–5900. https://doi.org/10.1523/JNEUROSCI.0740-06.2006 (2006).

49. Carmack, S. A. et al. Heroin addiction engages negative emotional learning brain circuits in rats. *J. Clin. Investig.* **129**, 2480–2484. https://doi.org/10.1172/jci125534 (2019).

50. Bravo, I. M. et al. Divergent behavioral responses in protracted opioid withdrawal in male and female C57BL/6J mice. *Eur. J. Neurosci.* **51**, 742–754. https://doi.org/10.1111/ejn.14580 (2020).

## Acknowledgements

We thank NIDA (NIH, USA), USA Drugs Supply program to provide morphine and animal care staffs. This work was supported by the University of Strasbourg initiative of Excellence (to ED), by Agence National de la recherche (ANR-23-CE16-0016, to ED),by Institut pour la Recherche en Santé Publique (SPAV1-22-018, to ED), by the National Institute of Health (# DA005010 and # DA048796 to BLK), and by the Canada Fund for Innovation, the Canada Research Chairs to ED and BLK.

## Author contributions

D.O., B.L.K. and E.D. designed the experiments. D.O. performed and analyzed the behavioural experiments. J.M. contributed to behavioural experiments for escalating procedure. D.O. and E.D. wrote the manuscript. B.L.K. edited the manuscript. All authors read and approved the submitted version.

## Competing interests

The authors declare no competing interests.

## Additional information

**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1038/s41598-024-60759-3.

**Correspondence** and requests for materials should be addressed to E.D.

**Reprints and permissions information** is available at www.nature.com/reprints.

**Publisher's note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
<figure><img src="image_47.png" /></figure>

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International

License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the
Creative Commons licence, and indicate if changes were made. The images or other third party material in this
article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the
material. If material is not included in the article's Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from
the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

© The Author(s) 2024