Long-lasting behavioral, molecular and functional connectivity alterations after chronic exposure to THC in adolescent mice

doi:10.21203/rs.3.rs-4830650/v1

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Long-lasting behavioral, molecular and functional connectivity alterations after chronic exposure to THC in adolescent mice

https://doi.org/10.21203/rs.3.rs-4830650/v1

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Heavy and daily use of cannabis with high contents of Δ⁹-tetrahydrocannabinol (THC) during adolescence is associated with an increased risk of developing psychotic disorders later in life. Here, we have generated a mouse model of THC exposure during adolescence that exhibits impairments in social interaction and increased vulnerability to develop sensorimotor gating deficiencies comparable to those previously described among heavy cannabis consumers. Importantly, we provide evidence on long-term cortico-striatal dysconnectivity induced by exposure to THC during adolescence and its correlation with impaired social interactions occurring later in adulthood. Moreover, we have observed long-lasting molecular alterations in key elements that regulate the mesolimbic dopaminergic system, namely on the balance between dopamine D₂, adenosine A_2A, and cannabinoid CB₁ receptors in the striatum of treated mice. Together, these findings contribute to a better understanding of the neurobiological bases of the deleterious effects associated with cannabis abuse during adolescence and point to the D₂R, A_2AR and CB₁R equilibrium as a potential target to reverse or prevent these effects.

Health sciences/Diseases/Psychiatric disorders/Addiction

Biological sciences/Neuroscience/Molecular neuroscience

Cannabis consumption has increased worldwide in the last years, likely due to the decrease in risk perception derived from the legislative changes regulating medical and/or recreational use in many countries¹. Epidemiological data reveal that heavy and daily use of cannabis with high content of the psychoactive compound Δ⁹-tetrahydrocannabinol (THC) is associated with an increased risk of developing a cannabis use disorder² and serious mental illness such as psychotic disorders³. Importantly, male adolescents are particularly vulnerable to experiencing the neuropsychiatric symptoms associated with cannabis abuse, including both positive (i.e., hallucinations and delusions) and negative (i.e., cognitive and sociability deficits) symptoms of psychotic disorders, as well as anxiety and other mood disorders^4–6. To date, no specific therapies have been developed to treat these adverse effects associated with cannabis abuse despite the increasing demand for specialized treatment for problems related to cannabis consumption⁷. To progress toward more specific and effective therapies to deal with this growing health problem, it is essential to better understand the neurobiological bases of such deleterious effects of cannabis abuse. The main objective of this study is to contribute to this challenge by analyzing the long-lasting behavioral, molecular and functional connectivity alterations in the brain induced by chronic exposure to THC in male and female adolescent and adult mice.

Current knowledge of the neurobiological bases of psychotic disorders indicates that the dysregulation of dopaminergic brain circuits plays a fundamental role in the development of psychotic symptoms⁸. Specifically, psychotic symptoms such as hallucinations and delusions are related to the hyperactivity of the mesolimbic dopaminergic pathway and the increased activity of the dopamine (DA) D₂ receptor (D₂R) in the striatum⁹. However, current data from human imaging studies and preclinical reports point to a disruption in multiple afferent systems that regulate dopaminergic neuron activity rather than to pathological changes within the DA system itself as the main alterations causing psychotic disorders¹⁰. In this sense, psychotic episodes induced by cannabis consumption with high THC content are concomitant with increased DA release in mesolimbic areas¹¹, which is mediated primarily by the THC-induced activation of CB₁R in the γ-aminobutyric acid (GABA)-ergic terminals projecting to dopaminergic neurons of the ventral tegmental area (VTA)¹². Furthermore, increasing evidence suggests that altered dopaminergic signaling underlying psychotic symptoms could be associated with adenosine deficiency in certain brain regions¹³. Different components of the adenosinergic system could contribute to the control of the mesolimbic dopaminergic system, but the strong antagonistic interaction between the adenosine A_2A receptor (A_2AR) and D₂R is known to play a key role^14,15. Thus, an imbalance between A_2AR and D₂R has been observed in striatal samples from preclinical models of psychotic-like behaviors and schizophrenic patients¹⁶. Interestingly, A_2AR also participates in the modulation of the THC effects on the dopaminergic system^17–19. Based on this evidence, the present study focusses first on the relationship between striatal levels of A_2AR, D₂R, and CB₁R as potential substrates underlying the harmful effects of chronic exposure to THC observed in adolescent and adult mice.

According to emerging theories holding that psychotic disorders result from the disruption of topological architecture in large-scale brain networks rather than from the lesion of isolated brain regions²⁰, we have also explored the potential alterations in the whole-brain connectome and cortico-striatal functional connectivity in THC-treated mice by using functional magnetic resonance imaging (fMRI) and graph theory as a powerful tool to accurately quantify the whole-brain connectivity network²¹ and its hypothetical correlation with aberrant behaviors later in adulthood.

Taken together, our findings contribute to a better understanding of the neurobiological bases of the long-lasting deleterious effects associated with cannabis abuse during adolescence.

Animals

Male and female C57BL/6J mice aged 4 weeks (adolescents) and 4 months (adults) were purchased from Janvier Labs (Le Genest-Saint-Isle, France). Animals were housed 3–4 per cage and maintained under standard animal housing conditions in a 12-h dark-light cycle with free access to food and water. Mice were randomly assigned to treatment groups and the experiments were conducted under blind experimental conditions. All animal procedures were carried out following the guidelines of the European Communities Council Directive 2010/63/EU and with the approval of the local ethics committee of the University of Barcelona (CEEA 417/18).

Pharmacological treatment

THC was purchased from Sigma-Aldrich Inc (St Louis, MO, USA) and dissolved in 5% ethanol, 5% Tween and 90% saline for intraperitoneal (i.p.) injection. Mice received a daily THC (5 mg/kg) or vehicle i.p. administration for 1 month. The volume administered was 10 mL/kg of body weight. According to the formula for dose translation based on body surface area²², the calculated human equivalent dose (HED) corresponds to 0.4 mg/kg of THC, which is approximately equivalent to the administration of 4 standard joint units²³ in a human being and resembles a heavy use of cannabis²⁴. Adolescent mice were treated with THC from post-natal day 28 (PND 28, early adolescence) to PND 60 (late adolescence). In the case of adult mice, they were treated with THC from PND 112 to PND 144.

Behavioral evaluation and sample collection

Behavioral testing started at PND 112 for the animals exposed to THC during adolescence and at PND 196 for those animals exposed to this drug during adulthood (Fig. 1A). Animals were consecutively evaluated in the elevated plus maze (EPM), the novel object recognition test (NORT), the three-chamber test, and the prepulse inhibition of the acoustic startle reflex test (PPI), as explained later. Five days after the end of the PPI test, the animals were subjected to resting state functional magnetic resonance imaging (rs-fMRI). Two days after the rs-fMRI, the animals were sacrificed, and their brains were rapidly dissected, frozen, and stored at – 80ºC until use.

Elevated Plus Maze (EPM)

Anxiety-like behavior was evaluated in the EPM, which consisted of a black plastic apparatus with four arms (15 x 5 cm) set in a cross from a neutral central square (5 x 5 cm) and elevated 40 cm above the floor. Two opposite arms were enclosed by vertical walls (closed arms), whereas the two other opposite arms had no protected edges (open arms). The total number of visits and the time spent in the open and closed arms were manually recorded through a closed-circuit camera system during the 5-min observation session.

Novel object recognition test (NORT)

Memory performance was assessed by NORT using a V-maze²⁵. Three hours after the end of the EPM test, the mice were placed within the V-maze (30 cm long x 5 cm wide each arm) containing two identical objects located at the ends of the maze arms. The time that each mouse spent exploring these objects was recorded through a closed-circuit camera system. Twenty-four hours later, the animals were reintroduced into the same V-maze, but this time one of the familiar objects was replaced by a novel one. To assess memory performance, an object discrimination index (DI) was calculated using the following formula: DI = (T_N − T_F)/(T_N + T_F), where T_N represents the time spent exploring the novel object, and T_F represents the time spent exploring the familiar object.

Three-chamber test

Social interaction and social memory of mice were evaluated in the three-chamber test one day after the end of NORT. The apparatus consists of a box (48 x 60 x 40 cm) divided into three chambers that are separated by two removable walls. First, the subject mouse was placed in the center of the middle chamber for 5 minutes as a habituation phase. Next, session 1 (sociability test) consisted of positioning a novel mouse (hereinafter referred to as mouse 1) inside a wire cage in one of the lateral chambers and a novel object (identical but empty wire cage) in the opposite one. The time spent exploring the novel mouse (mouse 1, T_M) or the novel object (T_O), was analyzed during 10 min. The social memory and novelty test (session 2) began immediately after the sociability test. In this test, the previous mouse (mouse 1) remained in its wire cage (now called the familiar mouse) and a novel mouse (mouse 2) was placed in the wire cage on the opposite side. Again, the time spent exploring the familiar mouse (mouse 1, T_F) or the novel mouse (mouse 2, T_N), was recorded manually for 10 min. Then, Sociability Index (SI) and Social Memory Index (SMI) were calculated, respectively, for the first and second sessions: SI = (T_M-T_O)/(T_M+T_O); SMI = (T_N-T_F)/(T_N+T_F).

Prepulse inhibition of the acoustic startle reflex test (PPI)

Three hours after the end of the three-chamber test, the PPI was performed to evaluate sensorimotor gating in mice, which is known to be impaired under psychotic conditions²⁶. Two automated StartFear combined system chambers (LE116, Panlab, Harvard Instruments, Spain) were used to test PPI in mice, as previously described¹⁶. Startle amplitude after prepulse (70, 75 and 80 dB) and pulse (120 dB) was automatically detected by PACKWIN V2.0 software. The percentage of prepulse inhibition (% PPI) for each prepulse intensity, was calculated as: %PPI = (startle amplitude on pulse alone – startle amplitude on prepulse trial)/(startle amplitude on pulse alone) x 100.

Resting-state Functional Magnetic Resonance Imaging (rs-fMRI)

Image acquisition

A 7.0T BioSpec 70/30 horizontal animal scanner (Bruker BioSpin, Ettlingen, Germany), equipped with an actively covered gradient structure (400 mT/m, 12 cm inner diameter) was used to scan all mice. Mice were placed in a Plexiglas holder and were fixed in a nose cone and exposed to a combination of anesthetic gases [medetomidine (bolus of 0.3 mg/kg, 0.6 mg/kg/h infusion) and isoflurane (0.5%)].

A 3D-localizer scan was used to ensure accurate position of the head at the isocenter of the magnet. T2-weighted image was obtained using a RARE sequence [effective TE = 33 ms, TR = 2.3 s, RARE factor = 8, voxel size = 0.08 x 0.08 mm2, slice thickness = 0.5 mm]. The acquisition of rs-fMRI was performed using an echo planar imaging (EPI) sequence [TR = 2 s, TE = 19.4, voxel size 0.21 x 0.21 mm2, slice thickness = 0.5 mm]; 420 volumes were acquired during 14 min.

rsfMRI: Image analysis and processing

Two approaches were used to evaluate functional connectivity, as previously described^27,28. On the one hand, whole brain connectivity was evaluated using global and regional network metrics²¹ and on the other hand a seed-based analysis was performed to evaluate connectivity of the nucleus accumbens (NAc) and Caudate Putamen (CPu) with the rest of the brain. For both approaches, rs-fMRI was preprocessed, including slice timing, motion correction by spatial realignment using SPM8, correction of EPI distortion by elastic registration to the T2-weighted volume using ANTs ²⁹, detrend, smoothing with a full-width half maximum (FWHM) of 0.6mm, frequency filtering of the time series between 0.01 and 0.1 Hz and regression by motion parameters. All these steps were performed using NiTime (http://nipy.org/nitime). Brain parcellation was performed by registration of a mouse brain atlas³⁰ to the T2-RARE acquisition of each subject using ANTs diffeomorphic registration. The region parcellation was then registered from the T2-weighted volume to the preprocessed mean rs-fMRI volume. The whole-brain functional brain network was estimated considering the grey matter regions obtained by parcellation as the network nodes. The connectivity between each pair of regions was estimated as the Fisher-z transform of the correlation between the average time series in each region. Network organization was quantified using regional and global graph metrics. Strength, local efficiency and clustering coefficient were calculated for every region, and the connectivity of the entire brain was quantified by the average strength, global and local efficiency, and the average clustering coefficient²¹. To perform the seed-based analysis, NAc and CPu were selected from the automatic parcellation. The average time series in the seed was computed and correlated with each voxel time series, resulting in a correlation map describing the connectivity of the striatum with the rest of the brain. The following regions of interest were identified from brain parcellation to evaluate their connectivity with the seed regions: (1) the cluster formed by the entorhinal (Ent), piriform (Pir), and insular cortex (Ins), (2) cingulate cortex (CgC), (3) hippocampus (Hipp), (4) medial prefrontal cortex (mPFC), (5) motor cortex, (6) sensory cortex, and parietal association cortex. Connectivity was quantified as the mean value of the correlation map in each region, considering only positive correlations.

Gel electrophoresis and immunoblotting

Striatal samples from mice were homogenized in ice-cold 10mM Tris HCl (pH 7.4), 1mM EDTA and 300mM KCl buffer containing a protease inhibitor cocktail (Roche Molecular Systems, USA). The homogenates were centrifuged for 10 min at 1000 × g. The resulting supernatants were centrifuged for 30 min at 12,000 × g and the pellets were resuspended in 50mM Tris HCl (pH 7.4) and 10 mM MgCl solution. Protein concentration was determined using the BCA protein assay kit (Thermo Fisher Scientific, Inc., Rockford, IL, USA) and equal amounts of protein (10 µg) for each sample were loaded and separated by electrophoresis on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (10%) gels. Proteins were transferred to Hybond®-LFP polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Chicago, IL, USA) using a Trans-Blot® SD semidry transfer cell (Bio-Rad, Hercules, CA, USA). Then, PVDF membranes were blocked with 5% non-fat milk in phosphate buffered saline (PBS) buffer containing 0.05% Tween-20 (PBS-T) for 45 min. After washing, membranes loaded with the striatal samples were incubated overnight at 4ºC with rabbit polyclonal anti-CB₁R (1:1500; Frontier Institute Co. Ltd., Shinko-nishi, Ishikari, Hokkaido, Japan), mouse monoclonal anti-A_2AR (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA, sc-32261), rabbit polyclonal anti-D₂R (1:1500; Frontier Institute Co. Ltd) and goat polyclonal anti-DAT (1:1000; Santa Cruz, sc-1433) antibodies in blocking solution overnight at 4°C. Protein loading was monitored using an antibody against beta-tubulin (1:10000, Abcam, Cambridge, UK). PVDF membranes were washed with PBS-T three times (5min each) before incubation with either a horseradish peroxidase (HRP)-conjugated goat antirabbit IgG (1/30000; Pierce Biotechnology, Rockford, IL, USA), HRP-conjugated rabbit anti-goat IgG (1/10000; Pierce Biotechnology) or HRP-conjugated goat anti-mouse (Thermo Fisher Scientific) in blocking solution at room temperature for 2 h. After washing the PVDF membranes with PBS-T 20 three times (5 min each), the immunoreactive bands were developed using a chemiluminescent detection kit (Thermo Fisher Scientific) and were detected with an Amersham Imager 600 (GE Healthcare Europe GmbH, Barcelona, Spain). Densitometric quantification was carried out with Image J (NIH, US). Bands were normalized to beta-tubulin.

Statistical analyses

Statistical analysis was performed with GraphPad Prism 9 (RRID:SCR_002798; San Diego, CA, USA). Statistical difference was set at p < 0.05. The number of samples/animals (n) in each group is indicated in the corresponding figure legend. Data normality was assessed using the Shapiro–Wilk test. Univariate outliers were assessed using the Grubbs’s test, while multivariate outliers were detected by the Mahalanobis distance method. Comparisons between experimental groups were performed by two-way ANOVA with treatment and sex as between factors (behavioral test results, immunoblotting quantifications and fMRI results), followed by Tukey’s post hoc when required. Pearson's correlation coefficients were calculated because all data was parametric.

Clustering analyses were performed using IBM SPSS Statistics v24.0 software (Systat Software Inc., Chicago, IL, USA). Two-step cluster analysis was used to classify the entire cohort of animals according to their higher/lower %PPI 80dB. Bayesian Criterion (BIC) was used to estimate of the maximum number of clusters, whereas the Euclidean method was used as a distance measure. To avoid bias in our data set, we did not predetermine the number of clusters. Chi-square analyses were performed to compare the percentage of mice in each cluster per treatment group. The effect size was calculated and expressed as the relative risk.

Behavioral effects of chronic exposure to THC in adolescent and adult mice

Chronic THC treatment during adolescence or adulthood did not produce any long-lasting alteration in the anxiety levels of male and female mice measured in the EPM (Fig. 1A). On the contrary, animals exposed to THC during adulthood, but not during adolescence, exhibited memory impairment in NORT later in life (Fig. 1B). Thus, two-way ANOVA revealed a significant effect of treatment in these animals and subsequent Tukey’s post hoc test confirmed that the significant decrease in the discrimination index was present in both, male (p < 0.01) and female (p < 0.001) animals chronically treated with THC during adulthood when compared to vehicle-treated mice.

Sociability and social memory deficits were evaluated in the three-chamber test. Two-way ANOVA revealed a treatment effect in animals exposed to THC during adolescence, but not during adulthood, and a tendency (p = 0.07) for the interaction between treatment and sex in the sociability index. Subsequent post hoc comparisons revealed that only male animals chronically treated with THC during adolescence showed a decreased sociability index when they reached adulthood (p < 0.001) (Fig. 1C). No significant changes in social memory were observed in any of the experimental groups (Fig. 1C).

Impairments of PPI, a sensorimotor gating process, are observed in patients with schizophrenia²⁶ and in patients suffering cannabis-induced psychotic symptoms³¹. In our experimental conditions, no significant PPI deficits were observed in any experimental group at any of the prepulse intensities evaluated, independently of the age when mice were exposed to THC or their sex (Fig. 1D shows results with 80 dB prepulse intensity). Similarly, no differences were observed between the groups in the startle response to any pulse intensity tested (data not shown). However, clustering analysis identified two populations of mice, one vulnerable (low %PPI) and another resilient (high %PPI) to sensorimotor gating deficits. Interestingly, a significantly higher proportion of the vulnerable population was found only in mice exposed to THC during adolescence (χ²₍₁₎ = 4.452, p < 0.05). Among these vulnerable mice treated with THC during adolescence, 67.7% were males, revealing a sex-dependent predisposition to develop sensorimotor gating impairment in adolescent mice exposed to this drug (Fig. 1E). Estimating the relative risk of developing sensorimotor gating impairment revealed that mice exposed to THC during adolescence, but not during adulthood, were 4.02 times more likely to belong to the vulnerable group (low %PPI 80 dB) than those exposed to vehicle (Fig. 1F). Statistical details of all the behavioral tests are included in Supplementary Table 1.

THC exposure during adolescence creates an unbalance between D₂R, CB₁R and A_2AR

Striatal dopamine dysfunction induced by THC is believed to be the basis for psychotic symptoms in cannabis users¹¹ and can be modulated by the adenosinergic system^17–19. Thus, we evaluated some key components of the dopaminergic, adenosinergic, and endocannabinoid systems in striatal samples of treated mice by immunoblotting (Fig. 2). Specifically, we determined the protein density of D₂R, A_2AR, CB₁R and the DA transporter (DAT). Two-way ANOVA revealed a significant effect of treatment during adulthood, but not during adolescence, in mice. However, Tukey’s post hoc test did not reveal significant differences between specific experimental groups (Fig. 2B). Statistical details of immunoblotting results are included in Supplementary Table 2.

As we hypothesized that an imbalance between these receptors could lead to psychotic-like alterations, we analyzed the correlations between their relative density. We observed a positive correlation between the density of D₂R and A_2AR, D₂R, and CB₁R, as well as A_2AR and CB₁R in striatal samples in vehicle-treated animals regardless of age at the beginning of treatment (statistics are detailed in Fig. 2C). Interestingly, these correlations disappeared in mice that were treated with THC during adolescence, except the CB₁R and A_2AR correlation that was preserved. Animals treated with THC during adulthood showed weaker correlations than those observed in vehicle-treated animals, but only the CB₁R-A_2AR correlation was significantly lower.

Long-term THC did not affect global brain network, but alters cortico-striatal connectivity in males exposed to THC during adolescence

Graph theory enables to quantify global network metrics and consequently to investigate the whole-brain network reorganization rather than isolated changes in the connectivity between specific brain areas²¹ and facilitates the characterization of the connectivity between a specific region and the rest of the brain³². In this study, we use graph theory to explore the impact of exposure to THC during adolescence and adulthood on both global and regional functional network metrics, focusing on the NAc and CPu networks. Weighted and binary measures were calculated for both hemispheres. Regarding global brain networks, no statistically significant differences in global efficiency, local efficiency, clustering coefficient, or strength between groups were found after FDR correction (Table 1). Similarly, when we target NAc or CPu, no statistically significant differences in efficiency, clustering coefficient, or strength between groups were found after FDR correction (Table 1).

Next, we evaluated the changes in the functional connectivity between specific brain areas induced by chronic treatment with THC during adolescence or adulthood and the potential correlation with the observed behavioral alterations. We performed a seed-based analysis of functional connectivity between NAc or CPu and selected brain regions previously described as involved in the development of psychotic-associated behaviors³³. Our results reveal alterations in the functional connectivity between left NAc and the left Enr, Pir, Ins cluster (treatment effect), the left (sex effect) and right (treatment effect) cingulate cortex, left (treatment and sex interaction) and right (sex effect) mPFC in the animals treated with THC during adolescence, but not during the adulthood. Subsequent post hoc analyses revealed a lower connectivity between the left NAc and the left Enr, Pir, Ins cluster (q < 0.01) and the left mPFC (q < 0.05) in male mice exposed to THC during adolescence compared to the vehicle control group. Moreover, a significant reduction in the functional connectivity between left NAc and the left CgC (q < 0.05) and left (q < 0.01) and right (q < 0.05) mPFC was found in female compared to male animals treated with vehicle during adolescence. No statistically significant differences were observed in functional connectivity between CPu and any region of interest in any case, despite a tendency to a reduction in functional connectivity of this seed area in animals treated during adolescence. Statistical details of this seed-based analysis of functional connectivity are included in Supplementary Tables 3 (treatment during adolescence) and 4 (treatment during adulthood).

Interestingly, the functional connectivity of the left CPu with the right CgC, the right Hipp, and the right mPFC, as well as the right CPu with the right and left CgC, the right and left Hipp, the right and left motor cortex, the left sensory cortex, and the parietal association were significantly correlated with the social interaction data (q < 0.05 in all cases), suggesting an anatomical substrate for the observed THC-induced alterations in social interaction in treated mice. Statistical details of the correlations between functional connectivity and behavior are included in Table 2.

Exposure to THC during adolescence has been suggested to cause significant long-lasting changes in brain that are associated to an increased risk of developing psychotic symptoms. However, these THC-mediated plastic changes during this critical window for brain maturation are still poorly understood. This study provides compelling evidence of long-lasting, sex- and age-dependent behavioral, molecular, and brain functional connectivity alterations induced by chronic exposure to THC in mice. Specifically, daily exposure to THC during adolescence induced sociability deficits and increased the risk of developing sensorimotor gating impairment in adult male mice. In addition, this chronic treatment with THC caused an imbalance between the striatal dopaminergic, cannabinoid, and adenosinergic systems later in adult mice. Importantly, exposure to THC during adolescence induced long-term cortico-striatal dysconnectivity, which was correlated with impaired social interactions occurring later in adulthood. Additionally, male and female mice chronically treated with THC during adulthood exhibited memory deficits later in life.

Interestingly, our results showing that chronic THC treatment reduced sociability during adolescence, but not during adulthood, in adult male mice agree with some previous preclinical studies addressing this issue^34,35, and mimic the sociability problems reported by chronic cannabis users³⁶. On the contrary, exposure to THC during adolescence in our experimental conditions did not influence memory performance later in life, despite previous findings associated exposure to adolescent THC with cognitive impairment in adult mice³⁷. However, this apparent discrepancy could be explained by the fact that we evaluated mice treated with THC during adolescence two months after the end of the treatment and previous reports demonstrated that cognitive impairment caused by a synthetic cannabinoid in adolescent rodents³⁸ and THC in humans³⁹ can be reversed by sustained abstinence^40,41. Interestingly, that long washout period was not enough to mitigate memory impairment in male and female mice treated with THC during adulthood. Although previous preclinical⁴² and clinical studies⁴³ suggested that chronic exposure to THC can also cause cognitive decline in adults, the present study is the first to report THC-induced memory alterations after such a prolonged period of abstinence in mice and warns about the detrimental chronic effects of recreational doses of THC during adulthood, which have been poorly investigated^44,45. Future research is planned to investigate the specific neurochemical and anatomical substrates underlying these cognitive impairments in adult mice chronically exposed to high doses of THC.

Sensorimotor gating deficits measured by the PPI of the startle reflex are present in patients²⁶ and animal models⁴⁶ of psychotic disorders including those related to cannabis consumption³¹, among other mental illnesses, and are considered to reflect alterations in the limbic-motor circuitry that regulates the capacity to properly filter out non-relevant sensory information from the external environment⁴⁷. Our study reports that THC administration during adolescence did not induce global sensorimotor gating deficits in adult mice, in line with some previous studies^48–50 but in contrast to some others^35,51,52. These contrasting findings could be explained by the fact that the PPI is very sensitive to a long list of variables, even within healthy populations⁴⁷, so slightly different experimental conditions could lead to apparently opposing results. However, a clustering analysis of our results revealed that two significantly different populations can be identified among mice based on their PPI response (i.e. high PPI vs low PPI, or resilient vs vulnerable to sensorimotor gating deficits, respectively). Remarkedly, our data reveal a significantly higher proportion of the vulnerable population among mice exposed to THC during adolescence, which was predominantly constituted by males, which correlates with a higher relative risk of developing that sensorimotor gating impairment of mice exposed to THC during adolescence, but not during adulthood. That results are in the same direction that epidemiological data reveal an association between daily cannabis use during adolescence and the incidence of psychotic disorder rates³, which reinforces the validity of our experimental model as a useful tool to explore the detrimental effects of cannabis abuse. The greater vulnerability to the deleterious effects of THC observed in male mice, which is similar to what occurs among cannabis users^4–6, could be related to the low circulating levels of estrogens in these animals. Indeed, these gonadal hormones are hypothesized to confer protection against psychotic symptoms and other neuropsychiatric signs associated with cannabis use⁵³.

Considering that psychotic symptoms in cannabis users have been associated with striatal dopamine dysfunction¹¹ and the already known role of the adenosinergic and endocannabinoid systems in the control of dopaminergic activity^12,13, we next explored some key components of these neurotransmitter systems as potential substrates underlying the deleterious effects of chronic exposure to THC observed in mice. Our most remarkable finding is that the expression of D₂R and A_2AR, D₂R and CB₁R, as well as A_2AR and CB₁R, is positively correlated in striatal samples in all experimental groups except in those mice that were treated with THC during adolescence, in which no significant correlations were identified except for levels of CB₁R and A_2AR. These results suggest that an accurate balance between D₂R and A_2AR/CB₁R expression could be instrumental in the precise regulation of striatal dopaminergic activity and that a perturbation of this equilibrium could underlie behavioral alterations. In this sense, previous findings revealed that deficiencies in the expression of D₂R⁵⁴, A_2AR⁵⁵ and CB₁R⁵⁶ lead to altered sociability abilities in mice, as observed in mice exposed to THC during adolescence in the present study. Similarly, an imbalance between A_2AR and D₂R has been observed in striatal samples from preclinical models exhibiting sensorimotor gating deficits and in schizophrenic patients¹⁶. These results point to the D₂R, A_2AR and CB₁R equilibrium as a potential target to reverse or prevent the behavioral alterations induced by cannabis abuse.

To our knowledge, this is the first study conducted in mice that explores the long-term consequences of chronic exposure to THC in functional brain connectivity. Previous studies addressing the consequences of exposure to THC during adolescence reported short-term functional connectivity alterations, mainly in the hippocampal circuitry, but did not follow those animals until adulthood⁵⁵. Our results revealed no differences between experimental groups in the global brain connectivity, but a significant cortico-striatal dysconnectivity induced by exposure to THC during adolescence. Similar functional connectivity deficits have previously been described in cannabis users⁵⁶ and schizophrenic patients³³. Specifically, these alterations were found in the connectivity between the NAc and mPFC, and between the NAc and the Ent, Pir, Ins clusters in the left hemisphere. Interestingly, these deficits were limited to the limbic cortices, which are innervated by mesocortical DA⁵⁷. An equivalent dysconnectivity between the NAc and mPFC regions was previously reported in patients diagnosed with schizophrenia who used cannabis^58,59. Notably, differences in functional connectivity between hemispheres were found, which could be related to the decreased number of fibers in the corpus callosum connecting both hemispheres previously reported to be associated with long-term cannabis use⁶⁰. Importantly, our data provide evidence of a significant correlation between social interaction data and functional connectivity between certain cortico-striatal regions and the hippocampus, suggesting that these circuitries could contribute to the THC-induced alterations in social interaction in adolescent mice. Additional research is required to fully establish the relationship between the functional connectivity alterations and the behavioral and molecular alterations described here in mice chronically treated with THC during adolescence.

Overall, the present study provides novel insights into the neurochemical and neuroanatomical bases of the deleterious effects associated with THC exposure during adolescence using a murine model that replicates some of the fundamental traits of the detrimental consequences of cannabis abuse. Our results support the hypothesis that chronic use of THC during adolescence can interfere with developmental processes occurring in the brain circuits critical for appropriate information processing in adulthood⁶¹. Targeting such processes could be an optimal strategy to reduce the risk of developing neuropsychiatric disorders associated with adolescent cannabis exposure.

Conflict of interest

The authors have nothing to disclose.

Funding

This study was supported by Plan Nacional sobre Drogas – Ministerio de Sanidad grant 2020I041 to EA and by Ministerio de Ciencia, Innovación y Universidades–Agencia Estatal de Investigación-FEDER funds/European Regional Development Fund – “a way to build Europe” grant PID2020-118511RB-I00 to FC. Founded by MCIN/AEI /10.13039/501100011033 “ESF Investing in your future”, grant FPU19/03142 to LGA and grant PRE2019-088153 to NSF.

Author contributions

LGA participated actively in the design of the study, performed all the experiments, analyzed the results and wrote the first draft of the manuscript. NSF contributed to the execution of the behavioral tests. GS and FV designed and processed the data of the fMRI experiments, interpreted the results and contributed to writing the manuscript. EA and FC conceived and designed the study, supervised the experiments and the analysis of the results and wrote the manuscript.

Acknowledgments

We thank Centres de Recerca de Catalunya (CERCA) Programme/Generalitat de Catalunya for IDIBELL institutional support and Maria de Maeztu MDM-2017-0729 to Institut de Neurociències, Universitat de Barcelona. We are also grateful to the CannaLatan network members (CYTED-Programa Iberoamericano de Ciencia y Tecnología para el Desarrollo) for the fruitful discussion about the results. Moreover, we thank the staff of the Experimental 7T magnetic resonance imaging (MRI) unit (IDIBAPS) for their technical support.

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Tables 1-2 are available in the Supplementary Files section.

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

Table1.xlsx
Table 1
Table2.xlsx
Table 2
SupplementalFigure1.tif
Supplementary Figure 1. Average functional connectivity of the left Caudate Putamen (CPu) with some of brain areas analyzed from the left hemisphere (intrahemispheric, left panels) and the right hemisphere (interhemispheric, right panels) are represented in mice exposed to THC or vehicle (VEH) during adolescence (A) and adulthood (B). Each point represents data from an individual mouse. Two-way ANOVA with treatment and sex as between factors per each age was performed. Data are mean ± SEM (n = 9-10/group). CgC, cingulate cortex; Ent, entorhinal cortex; Hipp, hippocampus; mPFC, medial prefrontal cortex; Pir, piriform cortex.
SupplementaryTable1.pdf
SupplementalTable2.xlsx
SupplementalTable3.xlsx
SupplementalTable4.xlsx

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Long-lasting behavioral, molecular and functional connectivity alterations after chronic exposure to THC in adolescent mice

Status:

Version 1

Abstract

Figures

Introduction

Material and Methods

Animals

Pharmacological treatment

Behavioral evaluation and sample collection

Elevated Plus Maze (EPM)

Novel object recognition test (NORT)

Three-chamber test

Prepulse inhibition of the acoustic startle reflex test (PPI)

Resting-state Functional Magnetic Resonance Imaging (rs-fMRI)

Image acquisition

rsfMRI: Image analysis and processing

Gel electrophoresis and immunoblotting

Statistical analyses

Results

Behavioral effects of chronic exposure to THC in adolescent and adult mice

THC exposure during adolescence creates an unbalance between D₂R, CB₁R and A_2AR

Discussion

Declarations

Conflict of interest

Funding

Author contributions

Acknowledgments

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1

Long-lasting behavioral, molecular and functional connectivity alterations after chronic exposure to THC in adolescent mice

Status:

Version 1

Abstract

Figures

Introduction

Material and Methods

Animals

Pharmacological treatment

Behavioral evaluation and sample collection

Elevated Plus Maze (EPM)

Novel object recognition test (NORT)

Three-chamber test

Prepulse inhibition of the acoustic startle reflex test (PPI)

Resting-state Functional Magnetic Resonance Imaging (rs-fMRI)

Image acquisition

rsfMRI: Image analysis and processing

Gel electrophoresis and immunoblotting

Statistical analyses

Results

Behavioral effects of chronic exposure to THC in adolescent and adult mice

THC exposure during adolescence creates an unbalance between D2R, CB1R and A2AR

Discussion

Declarations

Conflict of interest

Funding

Author contributions

Acknowledgments

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1

THC exposure during adolescence creates an unbalance between D₂R, CB₁R and A_2AR