Adolescent morphine exposure induced long-term cognitive impairment and prefrontal neurostructural abnormality in adulthood in mice

doi:10.21203/rs.3.rs-4438943/v1

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Adolescent morphine exposure induced long-term cognitive impairment and prefrontal neurostructural abnormality in adulthood in mice

https://doi.org/10.21203/rs.3.rs-4438943/v1

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Opioids abuse in adolescence is becoming a pressing public health issue. Although evidence indicates that adolescent opioid exposure contributes to persistent changes in brain development, its long-term consequences in adulthood are still unclear. Here, we established a mouse model of adolescent morphine exposure and used the 5-choice serial reaction time task (5‐CSRTT) combined with behavioral tests to evaluate changes in cognitive behaviors. We found that exposure to morphine during adolescence led to deficits in multidimensional cognitive functions in mice, including attention, information processing speed, and behavior inhibition. Notable, these impairments persisted into adulthood. Furthermore, the morphine-exposed mice exhibited decreased learning efficiency and spatial memory. Adolescent morphine exposure also induced significant and persistent morphological changes and synaptic abnormalities in medial prefrontal cortex (mPFC) neurons, which may be responsible for cognitive impairments in adulthood. Together, our study identifies adolescence as a critical period during which opioid exposure acts profoundly on cognitive development and emphasizes that the disruption of neurons by opioids in adolescence may link mPFC-associated cognitive impairments in adulthood.

Biological sciences/Neuroscience/Learning and memory/Long-term memory

Biological sciences/Neuroscience/Molecular neuroscience

adolescence

morphine

cognition

memory

medial prefrontal cortex

Opioid abuse is one of the major public health problems around the world [1]. According to the National Center for Drug Abuse Statistics (NCDAS), in the United States and Canada, a total of 5,360 apparent opioid toxicity deaths occurred and almost 72% of overdose deaths are the result of opioid overdose [2]. It is worth noting that substance use is typically initiated during adolescence, and opioids are among the most prevalent drugs in adolescents [3, 4]. However, most of the research on opioid abuse focuses on adults, while few studies explore its effects on the young people, leaving a barrier to the prevention and treatment of opioid use in young people.

Adolescence is a critical period of brain maturation and development of the nervous system, especially for the impulsive control and decision-making [5, 6]. Adolescents are vulnerable to impaired impulsive control and therefore are more likely to actively seek addictive drugs [7, 8]. Although evidence indicates that adolescent opioid exposure contributes to persistent changes in brain development, its long-term consequences for impulsive behavior in adulthood are still unclear. In addition to impulsivity, adolescent opioid abuse is commonly associated with a variety of long-lasting cognitive impairments [9, 10]. As previously documented, chronic morphine treatment in adolescent mice induces despair-like behaviors and deficits in social behaviors in adulthood [11]. Adolescent morphine exposure leads to persistent anxiety-like behaviors in female rats [12]. Sub-chronic and chronic exposure to morphine in adolescent mice induced impairment in spatial memory and altered neuronal synaptic transmission [13]. These evidences suggest that adolescent exposure to opioids may have critical influences on cognition, which highlights the need for in-depth research on the long-lasting effects of early-life opioid exposure.

The medial prefrontal cortex (mPFC) is one of the central hubs for cognitive functions, including attention [14], behavioral flexibility [15], and the execution of behavioral plans [16]. The mPFC not only plays a crucial role in cognition but is also an integral part of the reward system. From birth to early adulthood, mPFC neurons and circuits undergo critical changes in physiological properties, as well as in the strength of their connections with distal brain regions (including neuromodulatory centers and the reward-related mesolimbic system) [14]. However, due to the extended developmental period of mPFC, it is more vulnerable to damage during adolescence [15]. Many neurodevelopmental disorders that emerge during childhood and adolescence involve alterations in mPFC-dependent behaviors, including depression, anxiety, schizophrenia, autism, and impulsive disorders [17]. Previous studies suggest that repeated morphine exposure in adult mice induced complex cognitive impairment, accompanied by alternations in the synaptic ultrastructure of mPFC [18]. However, there is still a lack of convincing evidence and systematic preclinical research on the long-term effects of chronic morphine exposure during adolescence on cognitive functions in adults.

Clinical and preclinical studies have developed various methods to study cognitive function. The five-choice serial reaction time task (5-CSRTT) is a classic cognitive task that allows simultaneous evaluation of multiple cognitive modalities in rodents, including attention, response inhibition, cognitive flexibility, operant motivation, and information processing speed [19–21]. These changes in cognitive patterns are highly associated with different brain damage and neuropsychiatric conditions. Studies have shown that increased perceptual load in 5-CSRTT (i.e., increased/shortened inter-trial intervals and reduced stimulation durations) is more effective in distinguishing subtle cognitive differences [22]. Furthermore, rodents can be classified into early adolescence (postnatal day [PND] 21–34), late adolescence (PND 34–46), early adulthood (PND 60–90) and late adulthood (PND > 90) based on maternal behavior, social play behavior, and sexual interaction [23]. In this study, adolescent mice received chronic morphine treatment and changes in cognitive functions, locomotion, and memory capacity were assessed using 5-CSRTT and other behavioral tests. Moreover, we also investigated the changes in early and late adulthood of the mice to determine the long-term impact of adolescent morphine exposure. Taking into account the importance of mPFC in decision-making and impulse suppression, neuronal activity based on c-Fos expression and alterations in the synaptic and dendritic spine of mPFC neurons were examined.

Animals

Adolescent C57BL/6J mice (male, 18–22 g, 5 weeks old) were obtained from the Experimental Animal Center of Xi'an Jiaotong University. Mice were housed individually in a humidity- (50 ± 5%) and temperature-controlled (22 ± 3°C) room, with access to food and water ad libitum, except when animals were subjected to 5-CSRTT training. Mice were allowed to acclimatize for 5 days before the experiments. All training and testing sessions were conducted during the light phase (lights on from 7:00 to 19:00). The experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Xi’an Jiaotong University. All efforts were made to minimize the number of animals used and their suffering.

Adolescent morphine exposure

Morphine hydrochloride was purchased from the National Institute for Food and Drug Control (Beijing, China) and dissolved in 0.9% saline. Adolescent mice of PND 35 were randomly divided into the morphine group (Mor) and the saline control group (Sal). From PND 42, the Mor and Sal group intraperitoneally (i.p.) received morphine (10 mg/kg, twice daily) or equal volume of saline (10 mL/kg), respectively for 7 days. These mice were kept in their home cage with access to food and water ad libitum until adulthood (PND 60).

5-CSRTT

The effect of adolescent morphine exposure on cognitive function in adult mice was investigated using 5-CSRTT installations (Campden, UK) and ABET II software (ver. 2.18, Lafayette Instrument Co.). Three days before 5-CSRTT, the mice were subjected to restricted water access for 2 hours each day, and were stroked and scratched every day to adapt to the experimenters in advance. Mice were trained in 5-CSRTT as described in our previous study with minor modifications[24]. In summary, the 5-CSRTT procedure consisted of a training phase (sessions 1–18), a baseline phase (session 19) and a variable testing phase. In the training sessions, mice gradually learned to obtain a 5% sucrose reward by making a nosepoke in one of the five holes with random illumination. Once the mice reached stable performance in the baseline session (session 19), they were subjected to a series of variable tests with a high perceptual load. These variable tests include reduced stimulus duration (RSD), short inter-trial interval (SITI), long inter-trial interval (LITI), and fixed interval (10s ITI). To investigate the persistence of cognitive changes in adult mice, baseline and variable 5-CSRTT tests were performed again after 60 days. See the supplementary materials for details of the training and test protocol.

Open field test (OFT)

Mice were individually placed in a gray opaque plexiglas box (45 × 45 × 30 cm) under dim light (80 lux) and each test lasted 15 min. Any-maze software (Stoelting Company, USA) was used to record the movement trail and analyze the locomotor activity of mice. The total time spent in the central field (28 × 28 cm) was measured as an anxiety index.

Novel object recognition (NOR)

NOR was carried out to test the recognition memory for novel objects[25]. Three test sessions were included: habituation, training, and testing. In the habituation session, each mouse was individually habituated to the white plastic chamber (45 × 45 × 28 cm) for 15 min. In the training session (after 12 h), two identical objects (object A) were fixed to the middle of the left 1/3 and right 1/3 lines, respectively, allowing mice to explore the two objects freely for 5 min which was repeated 3 times at 15 min intervals. In the testing session (after 3 h), one of objects A was replaced with a new object B, and the mice re-examined for 5 min again. After a 24-hour retention interval, object A was replaced with a novel object C, and object B was replaced with object A. The mice were also placed in the same box to explore freely for 5 min. Exploration was defined as inquisitive activity directed toward the object, such as touching the object with the nose or limbs or sniffing the object. Recognition index = novel object exploration time/ (novel object exploration time + familiar object exploration time).

Y-maze

To assess spatial working memory of mice, Y-maze spontaneous alternation task was performed in a Y-shaped device composed of three gray opaque plastic arms (29.5 × 6 cm) converging at equal angles with a 14 cm high wall and interoperable with each other. Mice were allowed to freely explore all arms of the maze for 5 min and traced by ANY-maze software. Spontaneous alternation is defined as entries into all three arms on consecutive occasions (ABC, BCA, and CAB). Spontaneous alteration = correct choices / (total entries − 2) × 100%.

Barnes maze

The Barnes maze was used to assess spatial reference memory of mice[26]. The maze consists of a circular opaque grey platform (70 cm diameter) with 20 evenly-spaced holes (5 cm diameter) located in its perimeter and elevated 50 cm above the floor. An opaque escape tunnel is mounted underneath one hole while the remaining 19 holes are left empty. Since the natural preference of rodents for dark enclosures, the open spaces and bright light of the upper surface of the platform drive mice to find the target escape holes. Objects of various shapes and colors were placed around the Barnes maze as visual cues. The experiment was divided into a habituation phase, a training phase, and a probe test. In the habituation phase, each mouse was put in the center of the apparatus and was guided into the escape tunnel, staying for 3 min. In the training phase, each mouse was put in an opaque cone placed at the center for 5 s. Then, the cone was removed and the mouse was allowed to explore the maze until it enters the escape tunnel (registered as an escape). If the mouse failed to find the target hole within 3 min, it was gently directed to the escape tunnel and stayed for 30 s. Mice were trained twice a day with at least 6 h intervals. The training phase lasted for 5 d. The probe test was conducted 3 d later, the escape tunnel was removed and the mouse was allowed to explore the maze for 3 min. ANY-maze software was used to record the movement trail of mice. The following parameters were analyzed: latency for the first entry to the target hole (escape latency), total distance traveled (total path length), counts of exploring the target hole (correct nosepoke), percentage of time in the target quadrant (time in target quadrant %).

Immunofluorescent staining

Ninety minutes after the final 5-CSRTT test, mice were perfused intracardially with PBS (pH = 7.4) and 4% paraformaldehyde (PFA). The brains were post-fixed in 4% PFA overnight, equilibrated with 30% sucrose at 4°C and then cryosectioned (25 µm). Sections were permeabilized with 0.3% Triton X-100, blocked with 5% goat serum, and then incubated rabbit anti-c-Fos antibody, (1:400, Cell Signaling Technology, USA) overnight at 4 ^◦C. Following rinses, sections were incubated in Cy3–conjugated Affinity pure Goat Anti-Rabbit IgG(H + L) (1:200, Proteintech, China) for 3h, rinsed, and then mounted on slides. Images of identical portions of the mPFC were obtained using a fluorescence microscope (ZEISS Axioscan 7, Germany). All images were acquired under identical settings and parameters, and then exported to ImageJ for counting.

Electron microscopy

Electron microscopic studies were conducted to analyze the possible changes in the synaptic ultrastructure within mPFC. Mice were anesthetized using 10% chloral hydrate and perfused intracardially with saline and perfusion solution (2.5% glutaraldehyde and 4% paraformaldehyde in 0.1M PB). The brains were removed and post-fixed in the perfusion solution overnight at 4°C. mPFC were removed and dissected into 1.0 mm thick sections. This study followed the experimental procedures as detailed by Yin et al. [18]. The electron microscope images (magnification 300003) were captured using a Hitachi 7650 electron microscope operated at 80 kV. The synaptic density, width of the synaptic cleft, length and thickness of the postsynaptic density (PSD) were measured in both excitatory and inhibitory synapses. We used Image-Pro Plus software (Media Cybernetics, USA) for quantitative analysis. To ensure accuracy, we analyzed 20 random images per mouse. All electron microscope images were coded and randomized manually for a blind analysis conducted independently by two researchers.

Golgi staining

Golgi-Cox staining was conducted to examine potential changes in the morphology of dendritic spines within mPFC. The brains were initially stored in Golgi-Cox solution in the dark for 28 days, followed by storage in a 30% sucrose solution for 5 days. Coronal sections (150 mm thick) containing the mPFC were obtained using a vibratome. These sections were then stained with ammonium hydroxide for 30 minutes, treated with 20% ammonium hydroxide for an additional 30 minutes, and fixed in Kodak Film Fixer for 30 minutes. Subsequently, the sections underwent dehydration in ethanol and clearing in a xylene solution. To enhance the accuracy and quality of analyzing spine morphology, Z-stacks of Golgi-stained images were captured using a microscope equipped with a 100x oil immersion objective (ZEISS). These stacks were then flattened into a single layer using ImageJ software (Fiji version, https://imagej.net/software/fiji/). The Sholl Analysis tool in FIJI was employed to analyze the neuron morphology of the mPFC, as described in the reference [27].

Statistical analysis

Data was analyzed and plotted with GraphPad Prism 9.0 (GraphPad, La Jolla, CA). Unpaired Student's t test was used for comparisons between the two groups. Repeated measures (RM) two-way ANOVA was conducted using morphine/saline administration as the between-subjects factor and time as the within-subjects factor. Tukey’s or Sidak’s post hoc analysis was applied, when applicable, to correct for multiple comparisons. All data was presented as the means ± standard error of the mean (SEM). Statistical significance was *P < 0.05, **P < 0.01, ***P < 0.0001. Statistical details can be found in the supplementary material.

1. Effect of adolescent morphine exposure on 5-CSRTT training sessions

Figure 1A illustrates the timeline of morphine exposure in adolescent mice as well as the training and testing of 5-CSRTT in their adulthood. 5-CSRTT offers a valuable tool for efficiently assessing different dimensions of cognitive modalities. Accuracy and omission are an integrated measure of attention. Interestingly, as shown in Fig. 1B, Mor mice showed significantly decreased response accuracy (F _{1, 221} = 6.699, P < 0.05) and increased omission (F _{1, 224} = 5.775, P < 0.05) during session 9, demonstrating decreased learning efficiency in attention-related parameters during the initial training session. However, Sal and Mor mice showed similar response accuracy and omission during the later training sessions (session 10–18). Correct response latency represents a measure of processing speed. Mor mice showed significantly higher correct response latency (Fig. 1C; F _{1, 224} = 6.631, P < 0.05) during session 9, but in subsequent training sessions, there was no difference compared to the Sal group. Perseverative responses and time-out responses are considered indicators of compulsivity and reflect cognitive inflexibility. During session 9, Mor mice had more perseverative responses (Fig. 1D; F _{1, 216} = 15.63, P < 0.0001) and time-out responses (F _{1, 195} = 23.32, P < 0.0001) compared to Sal mice. Particularly, this difference in perseverative response continued to exist in subsequent training stages (Fig. 1D; F _{1, 432} = 9.822, P < 0.01). Premature response reflects loss of impulse control and disinhibition of inappropriate responding and is recognized as an indicator of impulsivity. There was no difference in premature response between Mor mice and Sal mice throughout the training sessions (Fig. 1E). In 5-CSRTT, the motivation of operant behaviors is sensitively indexed by the latency to collect rewards and completed numbers of trails. During session 9, Mor mice had less completed trials than Sal mice (Fig. 1F; F _{1, 224} = 5.283, P < 0.05). However, in the later training sessions, there were no significant differences in these two parameters between the two groups. These results revealed that Mor mice exhibited abnormalities in the early stages of 5-CSRTT learning, specifically manifested in decreased attention, processing speed, motivation in operant behavior, and increased compulsivity. However, these differences gradually diminished in subsequent training sessions.

2. Effect of adolescent morphine exposure on 5-CSRTT baseline performance

After mice achieved stable 5-CSRTT performance with 80% accuracy and 20% omission on two consecutive days, the stimulation duration (SD) was fixed at 0.8 seconds, marking the beginning of the baseline period (session 19). Apart from the RM two-way ANOVA with morphine and time as factors, the unpaired t-test was conducted separately using the results of the last 3 days of the baseline to identify stable differences in cognitive behavior during the baseline session. As shown in Fig. 2A, there was no significance in response accuracy between the two groups. The omission rate in the Mor group was significantly higher than in the Sal group throughout the entire baseline testing period (F _{1, 216} = 14.63, P < 0.0001), and this indicator showed statistical differences in the last 3 days (t ₈₆ = 2.040, P < 0.05). Although the RM two-way ANOVA did not reveal any differences, the t-test analysis of the last 3 days’ result for correct response latency showed significant increases in the Mor group (Fig. 2B; t ₈₈ = 2.493, P < 0.05). Furthermore, significant increases in perseverative response (Fig. 2C; F _{1, 199} = 16.21, P < 0.0001), time-out response (F _{1, 217} = 7.566, P < 0.01), and premature response (Fig. 2D; F _{1, 224} = 15.69, P < 0.0001) could be observed throughout the entire baseline testing period, as well as during the last 3 days (perseverative: t ₈₈ = 2.662, P < 0.01; time-out: t ₈₈ = 2.696, P < 0.01; premature: t ₈₈ = 2.999, P < 0.01). There was no significance in trials completed and reward collect latency between the two groups (Fig. 2E), suggesting unchanged motivation in operant behavior. These results indicated that morphine exposure during adolescence significantly affected compulsive and impulsive behaviors in mice. In addition, attention and processing speed were also partly affected.

3. Effect of adolescent morphine exposure on 5-CSRTT variable testing

In order to further probe any cognitive alterations in mice, we manipulated the execution difficulty of 5-CSRTT by varying ITI and SD to achieve a higher perceptual load. The main parameters of the variable tests are depicted in Fig. 3A. The performance of the mice under the variable conditions was analyzed with an unpaired t-test. Mor mice showed a decrease in response accuracy in the RSD (Fig. 3B; t ₂₂ = 2.373, P < 0.05) and 10s ITI tests (t ₂₂ = 2.366, P < 0.05), while their omission rates were significantly higher than Sal mice in the RSD (t ₂₂ = 3.331, P < 0.01), SITI (t ₂₂ = 2.268, P < 0.05) and LITI test (t ₂₂ = 3.535, P < 0.01), clearly suggesting an impairment in attention especially under reduced stimulus duration. The correct response latency of the Mor mice increased in the RSD (Fig. 3C; t ₂₂ = 3.71, P < 0.01), suggesting a reduction of processing speed under a heavy perceptual load. Compared to Sal mice, Mor mice had more perseverative responses in RSD (Fig. 3D; t ₂₂ = 2.51, P < 0.05), LITI (t ₂₂ = 2.185, P < 0.05), and 10s ITI (t ₂₂ = 2.682, P < 0.05) and more time-out responses in 10s ITI (t ₂₂ = 2.309, P < 0.05). Premature responses were significantly higher in the Mor group in the RSD (Fig. 3E; t ₂₂ = 3.02, P < 0.01), LITI (t ₂₂ = 2.583, P < 0.05) and 10s ITI test (t ₂₂ = 2.996, P < 0.01). The number of completed trials decreased in the LITI (Fig. 3F; t ₂₂ = 3.24, P < 0.01) and the 10s ITI (t ₂₂ = 2.215, P < 0.05), while the reward collect latency increased in the 10s ITI (t ₂₂ = 2.128, P < 0.05). Since a consistent increase in perseverative, time-out, and premature response was found in baseline and variable tests, it suggested that the cognitive flexibility of mice is venerable to morphine exposure during adolescence.

4. 5-CSRTT baseline and variable test after an extended period in adulthood

To investigate the persistence of cognitive changes in adult mice, baseline and variable 5-CSRTT tests were performed again after an extended period. The experiment timeline is illustrated in Fig. 4A. After undergoing the last 5-CSRTT variable tests, mice were placed in their home cage for 60 days (PND 107 to 167). Subsequently, we conducted 5-CSRTT baseline tests (session 19) in mice for 16 consecutive days and analyzed behavioral differences between the Mor and Sal groups using RM two-way ANOVA. We noticed that during the 16-day baseline testing period, the mice exhibited an obvious trend of adaptation. Therefore, we analyzed the data from the first 8 days and the last 8 days separately to better reflect the behavioral differences during the baseline testing period. In addition, the behavioral data of the mice in the last 3 days were pooled and analyzed using the unpaired Student’s t-test. The response accuracy of the Mor group decreased significantly in the first 8 days (Fig. 4B; F _{1, 223} = 7.783, P < 0.01). However, no differences were found in the last 8 days of baseline test. In comparison, the omission rate of the Mor group increased significantly in the first 8 days (F _{1, 223} = 25.81, P < 0.0001) and in the last 8 days (F _{1, 216} = 37.4, P < 0.0001), indicating the persistence of the impairment in attention. The reward collection latency in the Mor group increased significantly during the entire baseline testing period compared to the Sal groups (Fig. 4C; first 8 days: F _{1, 223} = 20.77, P < 0.0001; last 8 days: F _{1, 216} = 17.61, P < 0.0001). The Mor mice had significantly more perseverative responses than Sal mice throughout the baseline testing period (Fig. 4D; first 8 days: F _{1, 183} = 6.077, P < 0.05; last 8 days: F _{1, 176} = 7.053, P < 0.01). The time-out response in the Mor group increased significantly only in the first 8 days (F _{1, 184} = 10.13, P < 0.01), while no difference was found in the last 8 days of baseline test. Interestingly, we found that premature responses in the Mor group increased significantly in the last 8 days (Fig. 4E; F _{1, 208} = 12.03, P < 0.01), suggesting a persistence of impulsivity in adult Mor mice. No difference was found in total number of trials and reward collect latency, indicating unchanged motivation in the baseline test. In summary, these results suggest that adult Mor mice exhibited sustained attentional impairments and increased compulsivity and impulsivity under 5-CSRTT baseline test.

The 5-CSRTT variable tests were carried out after the baseline tests. The performance of the mice under the variable conditions was analyzed with an unpaired t-test. The Mor group showed a decreased response accuracy in the RSD (Fig. 5A; t ₂₂ = 2.933, P < 0.01) and 10s ITI test (t ₂₂ = 2.265, P < 0.05), as well as an increased omission rate in the RSD (t ₂₂ = 2.533, P < 0.05) and SITI test (t ₂₂ = 2.646, P < 0.05). The correct response latency was increased in the RSD and 10s ITI test (Fig. 5B; t ₂₂ = 2.912, P < 0.01; t ₂₂ = 2.307, P < 0.05, respectively), indicating decreased processing speed in the Mor mice. Furthermore, perseverative responses increased in the LITI test and 10s ITI test (Fig. 5C; t ₂₂ = 2.726, P < 0.05; t ₂₂ = 2.215, P < 0.05, respectively), while premature responses increased in RSD and 10s ITI (Fig. 5D; t ₂₂ = 2.764, P < 0.05; t ₂₂ = 2.863, P < 0.01, respectively), suggesting increased cognitive inflexibility in Mor mice. The number of completed trials decreased in the LITI test (Fig. 5E; t ₂₂ = 2.213, P < 0.05), suggesting partially decreased operant motivation in Mor mice. Together, these results of the variable tests further confirmed that exposure to morphine in adolescence induced a long-lasting impairment in attention in adult mice, while highlighting deficits in compulsivity and impulsivity as a conserved cognitive characteristic of 5-CSRTT at both the training and testing stages.

5. c-Fos expression and synaptic abnormalities in mPFC in adult mice

Following the last 5-CSRTT variable test, we performed c-Fos immunofluorescence staining to investigate whether mPFC neurons were differentially activated in mice with adolescent morphine exposure[28]. Representative images of c-Fos expression in the mPFC region are presented in Fig. 6A. We found a significant increase in the total number of c-Fos positive neurons per mm² in the mPFC region of Mor mice (Fig. 6B, t ₄₂ = 14.72, P < 0.0001), indicating that mPFC were substantially activated in these mice. Furthermore, the expression of c-Fos increased in the three mPFC subregions of Mor mice, and this difference was most significant in the PrL subregion (t ₄₂ = 18.38, P < 0.0001). Golgi staining was performed to examine the density of the dendritic spine and the branching of mPFC neurons. Representative images of the dendritic spins of mPFC neurons are shown in Fig. 6C. We found that the dendrites of the Mor group protrude a significantly lower density of spines than the Sal groups (Fig. 6D; t ₂₄ = 3.107, P < 0.01). Furthermore, we analyzed the complexity of dendritic branching patterns by Sholl analysis. A Sholl profile was manually obtained by plotting the number of dendrite intersections against the radial distance from the center of the soma at a distance of 10 to 110 µm (Fig. 6E). Based on these radial images, we found that Mor mice had sparser dendrites of mPFC neurons (Fig. 6E; F _{1, 220} = 48.46, P < 0.0001), significant from 20 to 50 µm distal from the cell body compared to Sal controls. This result suggested that the branch complexities of dendrites of mPFC neurons were reduced in the Mor group. Next, we analyzed the changes in synaptic ultrastructure of mPFC neurons by electron microscopy. Representative images of the ultrastructure of excitatory and inhibitory synapses are shown in Fig. 6F. The total number of excitatory synapses, as well as the thickness of the postsynaptic density (PSD), decreased in Mor mice (Fig. 6G; t ₁₃₀ = 5.843, P < 0.0001; t ₄₀₀ = 12.73, P < 0.0001, respectively). Similarly, the total number of inhibitory synapses was significantly decreased in Mor mice (Fig. 6H; t ₆₂ = 1.898, P < 0.05). These results indicated that adolescent morphine exposure induced significant and persistent morphological changes and synaptic abnormalities in mPFC neurons in adult mice, which may be responsible for the long-lasting cognitive impairments in 5-CSRTT.

6. Effects of adolescent morphine exposure on learning and memory in adult mice

The training and testing of 5-CSRTT rely on the conditional learning and memories of the animals. Furthermore, there is still controversy over the effects of chronic opioid exposure on different types of memory ability in animals[29–31]. Therefore, we investigated the effects of adolescent morphine exposure on the learning and memory abilities of mice in adulthood. The experimental timeline is shown in Fig. 7A. A separate cohort of mice was subjected to adolescent morphine exposure and was tested for locomotion, spatial working memory, and object recognition memory in adulthood (PND 60–66 and PND 126–130). Learning and retrieval of spatial reference memory was tested from PND 140–145 with the Barnes maze.

In the early adulthood stage of the mice (PND 60–66, Fig. 7B), there were no significant changes in OFT center time, while Mor mice showed lower total distances compared to the Sal group (t ₂₈ = 2.198, P < 0.05), suggesting decreased locomotor activities in early adulthood. The spontaneous alternation in the Y-maze test is a reliable measure of spatial working memory. No change in spontaneous alternation was found in Mor mice. We used NOR test to assess spatial object recognition memory. There was no difference in the discrimination index for the novel object between the Mor and Sal groups in the 3 h interval, while a significantly decreased discrimination index was found in the Mor group in the 24 h interval (t ₂₈ = 2.134, P < 0.05). Furthermore, after PND 126, no differences were found in the OFT, Y-maze, and NOR tests between the Mor and Sal groups in adulthood (Fig. 7C). These results indicate that the locomotor activity and recognition memory of Mor mice are temporarily impaired in early adulthood. The mice then underwent a 5-day Barnes maze training session to assess learning of spatial reference memory (Fig. 7D). Compared to Sal mice, Mor mice had increased escape latency and path length, and decreased correct nosepoke and target quadrant time % during training sessions, indicating lower efficiency in spatial reference memory formation in Mor mice. We then conducted a probe test to assess retrieval of spatial reference memory in these mice. Representative heat maps and track plots are shown in Fig. 7E. In the probe test, Mor mice showed significantly higher escape latency (Fig. 7F; t ₁₅ = 2.647, P < 0.05), longer path length (t ₁₅ = 2.258, P < 0.05), fewer correct nosepoke (t ₁₄ = 2.199, P < 0.05), and less time spend in target quadrant (t ₁₄ = 2.619, P < 0.05) compared to the Sal group. These results demonstrated that the retrieval of spatial reference memory and learning strategy were impaired in the Mor group.

In the present study, we demonstrated that exposure to morphine in adolescence induces long-lasting and complicated cognitive deficits in mice. In particular, these impairments persisted into adulthood well after drug exposure had subsided. The results of 5-CSRTT revealed that mice with adolescent morphine exposure exhibited persistent impairments in attention, information processing speed, and operant motivation, while the compulsivity and impulsivity were abnormally higher in adulthood. Furthermore, we found that the morphine-exposed group exhibited abnormalities in 5-CSRTT learning, specifically manifested in decreased attention, processing speed, and increased compulsivity. This finding is further supported by the Barnes maze test, where the Mor group required more effort during training to locate the escape hole. Our study also suggested that adolescent morphine exposure induced significant and persistent morphological changes and synaptic abnormalities in mPFC, which may be responsible for persistent cognitive impairments in adulthood.

Attention can be described as selective concentration on certain elements of the environment, while ignoring other less relevant perceptible information [32]. 5-CSRTT is widely used in rodents to measure attention by analyzing response accuracy and omission rates [19, 33]. Previous reports have indicated that morphine exposure affects attention in mice, but the specific period of time during which these alterations occur is controversial [18, 34, 35]. We showed that after an extended period of morphine withdrawal, the Mor group showed persistent impairments in attentional performance during the first 8 days of baseline training and subsequent variable tests. This result is consistent with previous findings that exposure to morphine in rats leads to higher rates of omission during adolescence [36]. Furthermore, we found that Mor mice exhibited persistent decreases in attention during the initial learning stage of 5-CSRTT. However, once the mice learned the procedure, there were no significant differences in attention between the morphine group and the saline group, suggesting that the transient decrease in attention in the Mor group is likely the result of decreased learning ability. In the 5-CSRTT variable tests, significant attentional impairments were also observed in the Mor group. These impairments occurred in early and late adulthood, further indicating that exposure to morphine during adolescence has long-lasting negative effects on attention in mice.

Information processing speed is an intrinsic basis for cognitive functions and reflects fundamental cognitive efficiency (i.e., the rate at which information accumulates towards a decision) [37]. Slowed processing speed may adversely affect memory, attention, executive functioning, and learning capacity, deficits that are commonly observed in conditions such as aging, attention deficit hyperactivity disorder (ADHD), and drug addiction [38–40]. Clinical studies showed that morphine withdrawal, compared to sustained morphine exposure, decreases information processing speed in humans [40]. Our study showed that information processing speed was reduced in the Mor group at session 9 and gradually returned to normal at the early stage of adulthood. After a 60-day extend period, the mice resumed 5-CSRTT training (session 19), and the information processing speed of the Mor group dropped precipitously, implying a notable decrease in cognitive efficiency in Mor mice. It is noteworthy that this result is highly consistent with the changes in attention, which partially confirmed the validity of the above hypothesis.

Operant motivation has been defined as the process whereby goal-directed activities are initiated and sustained [41]. Previous studies showed that opioid-dependent mice have increased motivational behavior [35]. However, it has been observed that for adolescent morphine-exposed mice, motivational proclivity remains unchanged after 30 days of withdrawal [36]. In 5-CSRTT, operant motivation of mice was evaluated by the total number of trials completed and the reward collection latency. Interestingly, Mor mice exhibited a decrease in the number of trials completed only during the initial learning stage of 5-CSRTT (session 9). However, no discrepancy was observed in the two metrics during the subsequent training phase, even after the extended 60-day period. Collectively, these results failed to prove that the operant motivation of morphine-treated mice had changed. It is more likely that the diminished learning efficiency of the Mor group mice led to a lower number of trials completed within 30 minutes during the initial learning stage. As the number of subsequent training sessions and days increased, this disparity gradually vanished, which is basically in agreement with previous studies [36]. In the variable tests, although there were slight differences in the number of trials completed and the reward collection latency between the LITI and 10s ITI, these differences could be attributed to an increase in task complexity and slower information processing in the Mor mice.

Impaired executive function has profound influences on the propensity for impulsivity and compulsivity. Impulsivity is the tendency to act prematurely despite negative consequences, which arises from dysregulation of inhibitory processes [42]. Compulsivity refers to persistent repetitive execution of learned behavior, resulting from maladaptive learning habits [43]. Impulsivity has been associated with the initial stages of addictive behavior, while compulsivity has been associated with the later stages of addiction, notably continuation and maintenance of addictive behavior [44]. Impairment in both impulsivity and compulsivity has been closely associated with the onset, maintenance, and relapsing nature of drug dependence. Clinical studies suggest that individuals using opioids (e.g., heroin and fentanyl) or those in withdrawal may experience dysregulated inhibitory control [45, 46]. In the study by Moazen P et al., exposure to morphine increased impulsivity only transiently in adults and young adults but over a longer period in adolescents [36]. In the present study, adolescent morphine-exposed mice exhibited a significant increase in impulsivity and compulsivity during the early and late stages of adulthood, which is in agreement with previous studies [47, 48]. In variable tests, in addition to an increase in perseverative and time-out responses, Mor mice exhibited a notable increase in premature responses in RSD, LITI, and 10s ITI. These results may suggest an increased susceptibility to dysregulated impulsivity and compulsivity in Mor mice, since variable tests prolonged/shortened ITI (including LITI, 10s ITI, and SITI) and reduced stimulus (RSD) simulated an increase in perceptual load. In summary, adolescent morphine exposure induced persistent impairments in inhibitory control that continue into late adulthood.

Previous research has indicated that chronic and intermittent exposure to morphine in adolescents leads to impaired spatial working memory in mice [13]. These findings are in line with our result that adolescent morphine exposure induced deficits in spatial working memory. It should be noted that our use of the Barnes maze contrasts with previous research using the Morris water maze [13]; nevertheless, the assessment metrics and underlying principles underpinning the two behavioral paradigms exhibit significant similarity. Since the mPFC is an important neocortical center for modulation of cognitive functions, including attention [49], behavioral inhibition [50], long-term memory network [51], we further investigated the morphological changes and synaptic structures of the mPFC in adolescent morphine-exposed mice. We showed that Mor mice have a decrease in dendritic spine density and dendritic branching density of PFC neurons, which may be linked to persistent cognitive impairments in adulthood. Currently, there is limited study on the ultrastructural changes in PFC neurons after long-term withdrawal from adolescent morphine exposure. Through transmission electron microscopy, we observed a decrease in excitatory synaptic density, excitatory postsynaptic density width, and inhibitory synaptic density in adult PFC neurons from mice exposed to morphine during adolescence. In adolescent rats, excessive elimination of synapses in prefrontal neurons is associated with neuropathological changes [52]. Drugs or injuries in adolescent mice induce PFC neuron-specific defects, leading to structural damage to dendritic complexity and synaptic quantity [53]. These evidences are consistent with our findings. Together, our findings suggested that impaired synaptic structures of mPFC neurons in adolescence may link mPFC-associated cognitive impairments in adulthood.

Our research revealed that morphine exposure during adolescence leads to deficits in multidimensional cognitive functions in mice, including attention, information processing speed, and behavior inhibition. Specifically, these impairments persisted into adulthood. Furthermore, the morphine-exposed mice exhibited decreased learning efficiency and spatial memory. Adolescent morphine exposure also induced significant and persistent morphological changes and synaptic abnormalities in mPFC neurons, which may be responsible for the cognitive impairments in adulthood. Together, our study identifies adolescence as a critical period during which opioid exposure profoundly affects cognitive development and emphasizes that disruption of neurons by opioids in adolescence may link mPFC-associated cognitive impairments in adulthood.

Conflict of Interest

The authors declare no conflict of interests.

Acknowledgments

This work was supported by the Basic Science Research Program of Shaanxi Province (2021JM-027, 2022JQ-783) and the Natural Science Foundation of China (82070546).

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The authors have declared there is NO conflict of interest to disclose

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Adolescent morphine exposure induced long-term cognitive impairment and prefrontal neurostructural abnormality in adulthood in mice

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Abstract

Figures

INTRODUCTION

MATERIALS AND METHODS

Animals

Adolescent morphine exposure

5-CSRTT

Open field test (OFT)

Novel object recognition (NOR)

Y-maze

Barnes maze

Immunofluorescent staining

Electron microscopy

Golgi staining

Statistical analysis

RESULTS

1. Effect of adolescent morphine exposure on 5-CSRTT training sessions

2. Effect of adolescent morphine exposure on 5-CSRTT baseline performance

3. Effect of adolescent morphine exposure on 5-CSRTT variable testing

4. 5-CSRTT baseline and variable test after an extended period in adulthood

5. c-Fos expression and synaptic abnormalities in mPFC in adult mice

6. Effects of adolescent morphine exposure on learning and memory in adult mice

DISCUSSION

CONCLUSION

Declarations

Conflict of Interest

Acknowledgments

References

Additional Declarations

Supplementary Files

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