Adolescent Exposure to WIN 55212-2 Render the Nigrostriatal Dopaminergic Pathway Activated During Adulthood
Department of Pharmacy and Interdisciplinary Center of Neuroscience, Faculty of Chemistry and Pharmacy, Pontificia Universidad Católica de Chile, Santiago, Chile
Departments of Neuroscience, Psychiatry, and Psychology, Center for Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania
Department of Cellular and Molecular Biology, Faculty of Biological Science, Pontificia Universidad Católica de Chile, Santiago, Chile
Correspondence: José Antonio Fuentealba, PhD, Department of Pharmacy, Faculty of Chemistry and Pharmacy, Pontificia Universidad Católica de Chile, Santiago, Chile (jfuentea@uc.cl).Abstract
Background
During adolescence, neuronal circuits exhibit plasticity in response to physiological changes and to adapt to environmental events. Nigrostriatal dopaminergic pathways are in constant flux during development. Evidence suggests a relationship between early use of cannabinoids and psychiatric disorders characterized by altered dopaminergic systems, such as schizophrenia and addiction. However, the impact of adolescent exposure to cannabinoids on nigrostriatal dopaminergic pathways in adulthood remains unclear. The aim of this research was to determine the effects of repeated activation of cannabinoid receptors during adolescence on dopaminergic activity of nigrostriatal pathways and the mechanisms underlying this impact during adulthood.
Methods
Male Sprague-Dawley rats were treated with 1.2 mg/kg WIN 55212-2 daily from postnatal day 40 to 65. Then no-net flux microdialysis of dopamine in the dorsolateral striatum, electrophysiological recording of dopaminergic neuronal activity, and microdialysis measures of gamma-aminobutyric acid (GABA) and glutamate in substantia nigra par compacta were carried out during adulthood (postnatal days 72–78).
Results
Repeated activation of cannabinoid receptors during adolescence increased the release of dopamine in dorsolateral striatum accompanied by increased population activity of dopamine neurons and decreased extracellular GABA levels in substantia nigra par compacta in adulthood. Furthermore, perfusion of bicuculline, a GABAa antagonist, into the ventral pallidum reversed the increased dopamine neuron population activity in substantia nigra par compacta induced by adolescent cannabinoid exposure.
Conclusions
These results suggest that adolescent exposure to cannabinoid agonists produces disinhibition of nigrostriatal dopamine transmission during adulthood mediated by decreased GABAergic input from the ventral pallidum.
Boxed Text
Cannabis intake in teenagers is higher than in the adult population in Europe and the Americas, which results in a higher activation of CB1/2 receptors during adolescence. Furthermore, it has been observed that nigrostriatal dopaminergic pathway is maturing during adolescence. However, the consequences of adolescent activation of CB1/2 receptor in the adult nigrostriatal dopaminergic pathway remain to be addressed. Using neurochemistry detection and electrophysiological recordings, we observed a disinhibition of nigrostriatal dopaminergic transmission in adult rats after a repeated activation of CB1/2 receptor during adolescence. This result and previous evidence suggest that the activated state of dopaminergic nigrostriatal induced by adolescent cannabinoid exposure may be a risk factor that strongly predisposes to major neuropsychiatric disease later in life.
Introduction
Adolescence is characterized by a higher sensitivity to reward and an increase in risk-taking behaviors compared with adulthood (Sturman and Moghaddam, 2011; Doremus-Fitzwater and Spear, 2016). Consequently, this period is associated with the initiation of intake of drugs of abuse such as ethanol, nicotine, and cannabis (Doremus-Fitzwater and Spear, 2016; UNODC, 2018). In the case of cannabis, the annual prevalence of use among the adolescent population is higher than the general population in Europe and the Americas (UNODC, 2018). Furthermore, approximately 17% of those who initiate use of cannabis during adolescence develop cannabis use disorders; in contrast, just 9% who experiment with cannabis in adulthood develop cannabis use disorders (Lopez-Quintero et al., 2011; Volkow et al., 2014). However, the consequences of adolescent exposure to cannabinoids on neurobiological substrates associated with addiction remain unclear.
Dopaminergic transmission in the mesolimbic and nigrostriatal pathways is associated with the development of addictive behavior. Whereas the initial seeking of drugs of abuse is accompanied by an increase in dopaminergic mesolimbic activity (Everitt and Robbins, 2016; Robinson et al., 2016), the transition to a compulsive drug-seeking habit is associated with a progressive increase in dopamine neurotransmission in the nigrostriatal dopamine pathway (Ito et al., 2002; Willuhn et al., 2012; Everitt and Robbins, 2013). The activity of mesolimbic and nigrostriatal dopamine neurons is driven by different inputs (McFarland and Kalivas, 2001; Koob and Volkow, 2016), controlling extracellular dopamine levels in the nucleus accumbens (NAc) and dorsolateral striatum (DLS), respectively (Floresco et al., 2003; Panin et al., 2012). Neuroanatomical evidence has shown that substantia nigra pars compacta (SNc) receives gamma-aminobutyric acid (GABA)ergic input from multiple regions, such as striatum, external globus pallidum, substantia nigra pars reticulata (SNr), and ventral pallidum (VP), while the subthalamic nucleus and pedunculopontine nucleus drive their glutamatergic input on SNc (Watabe-Uchida et al., 2012; Steiner and Tseng, 2017). Of note, a dysfunction in the activity of these inputs can contribute to addictive-like behavior. For instance, inactivation of both subthalamic nucleus or pedunculopontine nucleus decreases drug-seeking of cocaine in adult rats (Corrigall et al., 2002; Baunez et al., 2005), while an imbalance in the activity of striatal medium spiny neurons increases drug-seeking behavior (Yager et al., 2015). Moreover, activation and inhibition of VP GABAergic neurons promote and reduce drug seeking of cocaine, respectively (Root et al., 2015; Farrell et al., 2019; Heinsbroek et al., 2020). Together, these studies suggest that the dysfunction of the nigrostriatal pathway induced by drugs of abuse (Willuhn et al., 2012) is accompanied by changes in the GABAergic and glutamatergic levels in SNc.
Cannabinoids play a role in multiple physiological processes, such as appetite, mood, memory, and motivation (Mechoulam and Parker, 2013; Pertwee, 2014). The CB1 receptors are crucial to synaptic communication due to their ability to modulate the release of different neurotransmitters, such as GABA, glutamate, and dopamine (Heifets and Castillo, 2009; Kano et al., 2009; Castillo et al., 2012). This receptor is widely expressed in the brain, highlighting regions such as prefrontal cortex, globus pallidum, VP, hippocampus, striatum, ventral tegmental area (VTA), and substantia nigra (Tsou et al., 1998; Egertová and Elphick, 2000; Mátyás et al., 2006, 2008; Davis et al., 2018). In VTA and substantia nigra, the CB1 receptor is mainly expressed in the axonal terminal region of GABAergic and glutamatergic projections, suggesting an indirect control of dopaminergic transmission (Julian et al., 2003; Yanovsky et al., 2003; Mátyás et al., 2006; Covey et al., 2017; Davis et al., 2018). It has been observed that an acute systemic administration of WIN 55212-2, a CB1/2 agonist, increases dopamine extracellular levels in the NAc (Tanda et al., 1997) and DLS (Polissidis et al., 2014) associated with an increase in firing rate of dopamine neurons in the VTA and SNc, respectively (French et al., 1997). This increase in the dopaminergic transmission induced by cannabinoids has also been observed after repeated adolescent exposure. An increase in the firing rate and burst activity of dopamine neurons from VTA is induced by adolescent exposure to Δ 9 tetrahydrocannabinol (Δ 9-THC), the psychoactive compound of cannabis and partial agonist of CB1/2 receptors (Renard et al., 2017). In addition, Gomes et al. (2015) showed that adolescent exposure to WIN 55212-2 increases the spontaneous activity of dopaminergic neurons of the VTA in adult rats (Gomes et al., 2015). While it has been suggested that adolescent use of cannabis is a risk factor in developing drug addiction in adults (Schneider, 2008), the studies of consequences of adolescent exposure to cannabinoid have been mainly concerned with the dopaminergic mesolimbic pathway (Pistis et al., 2004; Schneider, 2008; Higuera-Matas et al., 2010; Gomes et al., 2015; Renard et al., 2017).
The aim of this research is to assess the consequences of early exposure to cannabinoid on the nigrostriatal dopaminergic pathway in adulthood. We hypothesized that repeated activation of CB1/2 receptors during adolescence would activate the nigrostriatal dopaminergic pathway in adulthood. Microdialysis and single-unit recording experiments showed that adolescent exposure to WIN 55212-2 increases dopamine extracellular levels in DLS and decreases GABA extracellular levels in SNc accompanied by an increase in the population activity of dopamine neurons.
METHODS
All procedures were carried out accordance with the guidelines published in the NIH Guide for the Care and Use of Laboratory Animals (8th edition) and the principles presented in the “Guidelines for the Use of Animals in Neuroscience Research” by the Society for Neuroscience. The microdialysis protocols were approved by the local bioethics committee, verifying that it complies with the basic principles set forth in Chilean Law 20.380 on Animal Protection 2009 (ID project: 160816013). Electrophysiological experiments were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh.
Animals
In microdialysis experiments, adolescent male Sprague-Dawley (post-natal day [PD] 33) rats were obtained from the Animal Care Faculty of the Biological Sciences, Pontificia Universidad Católica de Chile (Charles River, Wilmington, MA; RRID: RGD_728193) under veterinarian supervision. Rats were maintained in Animal Care of the Department of Pharmacy, Pontificia Universidad Católica de Chile. In electrophysiological experiments, adolescent male Sprague-Dawley rats were obtained from Envigo (Indianapolis, IN). All rats were housed in groups of 2 per cage and kept at room temperature between 22°C and 24°C on a 12-hour-light/-dark cycle (lights on at 7:00 am EST) with access to food and water ad libitum. Rats were handled for 1 week before starting the treatment. A total of 75 adolescent rats were divided into 2 treatment groups: 38 rats for vehicle treatment and 37 rats for WIN 55212-2 treatment. All vehicle and treatment protocols were performed in parallel.
Reagents
The CB1/2 agonist, WIN 55212-2 mesylate (CAS no. 131543-23-2), was emulsified in 2% Tween 80 (CAS no. 9005-65-6) in saline solution 0.9% at a concentration of 1.2 mg/mL. The GABAa antagonist bicuculline methyl bromide (CAS no. 66016-70-4) (0.1 µg) was mixed fresh in Dulbecco’s buffer (MDL no. MFCD00131855) before starting recording. Reagents were obtained from Sigma-Aldrich (St. Louis, MO).
Treatment
Adolescent rats were daily injected i.p. with WIN 55212-2 (CB1/2 agonist) at a dose of 1.2 mg/kg (WIN) or with vehicle (2% Tween 80 in saline solution 0.9%) at a volume of 1 mL/kg from PD 40 to PD 65, similar to previously described (Gomes et al., 2015). Previous evidence has shown that cannabinoid exposure during this range of age modifies behavioral tests, such as object recognition, social interaction, and amphetamine-induced locomotion in adult rats (Schneider and Koch, 2003, 2005; Gomes et al., 2015). All the experiments were carried out between PD 72 and PD 78 (Figure 1).
No-Net Flux Microdialysis
To assess the effects of adolescent WIN 55212-2 exposure in nigrostriatal dopaminergic transmission, adult rats were anesthetized with urethane 1.5 g/kg i.p., and no-net flux microdialysis experiments in DLS were carried out as described in previous publications (Azocar et al., 2019; Pérez-Valenzuela et al., 2019). Urethane was chosen due to the extended half-life (Gumbleton and Benet, 1991). In addition, urethane does not modify basal and stimulated dopamine dialysate in striatum (Tepper et al., 1991; Howard and Feigenbaum, 1997). Anesthetized rats were placed in a stereotaxic apparatus, the skull was exposed, and a hole was drilled. A concentric microdialysis probe (CMA 11 Microdialysis, Holliston, MA) with an in vitro recovery rate higher than 10% was lowered vertically into the DLS using the coordinates +1.2 AP, +3.6 ML relative to bregma, and −4.8 DV from the dura (Paxinos and Watson, 2009). Body temperature was maintained by a thermostatically controlled electric heating pad. The probe was perfused for 40 minutes with Krebs–Ringer phosphate buffer with 0.2 mM acid ascorbic (AA-KRP) using a Harvard infusion pump (Harvard Apparatus, Holliston, MA) at a rate of 2 µL/min to allow equilibration. The composition of the AA-KRP was 120 mM NaCl, 2.4 mM KCl, 1.2 mM CaCl2, 0.9 mM NaH2PO4, 1.4 mM Na2HPO4, and 0.2 mM of ascorbic acid (pH 7.4). The probe was then randomly perfused with 5 different concentrations of dopamine—0.0, 5.0, 10.0, 20.0, and 40.0 nM—in AA-KRP to determine dopamine basal dialysate, dopamine extracellular concentration (Cext), and extraction fraction (Ed), an indirect measure of dopamine uptake (Smith and Justice, 1994). After a stabilization period of 20 minutes, 3 consecutive samples were collected every 5 minutes for each concentration of dopamine. Perfusion samples were collected in 2 µL of perchloric acid (0.2 N) and maintained on ice (4°C) until quantification.
Conventional Microdialysis
Conventional microdialysis experiments were carried out in SNc to determine basal dialysate levels of glutamate and GABA. Adult rats were anesthetized with urethane 1.5 g/kg i.p. and placed in a stereotaxic apparatus, the skull was exposed, and a hole was drilled. Then, a concentric microdialysis probe (CMA 11 Microdialysis, Holliston, MA) with an in vitro recovery rate higher than 10% was lowered diagonally into the SNc using a 40° angle from the horizontal axis at the following coordinates: −4.9 AP, +7.6 ML relative to bregma, and −8.0 DV from the dura (Paxinos and Watson, 2009). Body temperature was maintained by a thermostatically controlled electric heating pad. The probe was perfused for 40 minutes with KRP buffer at a rate of 2 µL/min using a Harvard infusion pump (Harvard Apparatus). After a stabilization period using KRP, 3 consecutive samples were collected every 5 minutes for the determination of GABA and glutamate basal dialysate. Perfusion samples were collected in 2 µL perchloric acid (0.2 N) and maintained on ice (4°C) until quantification.
Analysis of Dialysate Samples
Dopamine was quantified using a high-performance-liquid-chromatography (HPLC) system with an amperometric detector as described previously (Escobar et al., 2012). Twelve (10 µL of sample plus 2 µL PCA 0.2 N) µL of the collected samples was injected into an HPLC system (BASi America, West Lafayette, IN) with the following configuration: a pump (Jasco LC-Net II/ADC, Tokyo, Japan), a UNIJET LC column (part no. MF-8954, BASi), and an amperometric detector (LC4C, BASi America). The mobile phase contained 100 mM NaH2PO4, 1.0 mM ethylenediaminetetraacetic acid, 1.0 mM octane-1-sulfonic acid sodium salt, and 5% acetonitrile (pH 3.0), and it was pumped at a flow rate of 700 µL/min. The potential of the amperometric detector was set at 650 mV. Under these experimental conditions, the retention time for dopamine was 6 minutes.
Glutamate and GABA were quantified using an HPLC system with a fluorescence detector as described previously (Sotomayor-Zárate et al., 2010). Briefly, 12 μL of the sample of dialysis perfusate and PCA were mixed with 12 μL KRP and 4 μL borate buffer (pH 10.8), and then the mixture was derivatized by adding 4μL fluorogenic reagent (20 mg orthophthaldehyde and 10μL β-mercaptoethanol in 5mL ethanol). At 90 seconds after derivatization, samples were injected into an HPLC system with the following configuration: quaternary gradient pump (Jasco Co. Ltd.), a C-18 reverse phase column (Kromasil, Eka Chemicals, Bohus, Sweden), and a fluorescence detector (Jasco Co. Ltd.). A mobile phase containing 0.1 M NaH2PO4 and 23% CH3CN (pH 5.7) was pumped for 20 minutes. The flow rate of the mobile phase was set at 1.2 mL/min, and the retention time for glutamate was 3 minutes and for GABA was 16 minutes.
Single-Unit Recording
To assess the effects of adolescent WIN 55212-2 exposure on nigrostriatal dopaminergic transmission, single-unit recordings in SNc were carried out after at least a week of treatment. Adult rats were deeply anesthetized with chloral hydrate (400 mg/kg i.p.) for a non-survival procedure (Field et al., 1993). The anesthesia was monitored by assessing the foot pinch reflex and maintained throughout the experiment with additional doses of chloral hydrate (approximately 50 mg/kg, i.p.). Chloral hydrate was used in the recording experiments since it does not modify the percentage of spikes per bursts in bursting dopamine neurons in the SNc (Kelland et al., 1990). Anesthetized rats were placed in a stereotaxic apparatus (Stoelting), and the body temperature was maintained by a thermostatically controlled electric heating pad. A hole was drilled in the coordinates AP −4.9, ML +2.2 from bregma. The electrodes were pulled from Omegadot 2.0-mm glass tubing on a Narishige P-5 vertical electrode puller and the tip has broken back under microscopic control and filled with 2 M NaCl containing 2% Pontamine Sky Blue dye. The impedance of the electrodes was tested in situ ranging from 6 to 15 MΩ. The recording procedure was based on (Gomes et al., 2015). Six vertical tracks separated by 200 mm were sampled at the following coordinates: AP −4.9 to −5.1, ML 2.0 to 2.4 from bregma, and −6.5 to n−9.0 DV from brain surface (see Figure 2c). Single-unit activity was filtered using a high-pass filter at 30 Hz and low-pass at 10 kHz. Only stable spontaneous neuronal activity (at least 1–3 minutes) with a signal-to-noise ratio greater than 3:1 was analyzed. Dopamine neurons were identified according to the following criteria: (1) location; (2) an action potential duration >2.2 ms; (3) slow firing rate (<10 Hz); and (4) irregular and burst firing patterns, with the start of burst characterized by an interspike interval <80 ms and burst termination defined as a subsequent inter-spike interval >160 ms (Grace and Bunney, 1983; Ungless and Grace, 2012). At the end of the experiment, the electrode placement was marked using electrophoretic ejection of Pontamine Sky Blue dye from the tip of the electrode (20 μA constant negative current, 30 minutes).
Pharmacological Manipulations
To study the mechanism underlying the effects of adolescent exposure to WIN 55212-2 on nigrostriatal dopaminergic transmission, bicuculline methyl bromide (0.1 µg) was mixed fresh in 0.5 µL Dulbecco’s buffer and infused into the VP through a 30-gauge injection cannula protruding 2.0 mm past the end of the guide at an injection volume of 0.1 µL every 1 minute. The cannula was lowered to the following coordinate: AP −0.7, ML +2.9 from bregma, DV −6.0 from the skull (Paxinos and Watson, 2009). Ten minutes after the perfusion of bicuculline, a single-unit recording was carried out. The doses used in this study have been shown to not alter the basal activity of dopamine neuron (Floresco et al., 2003).
Histology
Rats were decapitated under deep anesthesia (urethane 1.5 g/kg i.p. in microdialysis experiments and chloral hydrate 400 mg/kg i.p. in single-unit recording), and brains were extracted and cleaned with NaCl 0.9%. Brains were stored in 4% paraformaldehyde. At least 2 days before slicing, the brains were cryoprotected using a solution of 30% sucrose. To assess the location of the probe and electrode, brains were frozen and sliced coronally into 50-mm-thick sections. Slices were stained with cresyl violet, and the probe/electrode placement was localized using the atlas of brain rat (Paxinos and Watson, 2009). Only data from correct probe/electrode placements were subjected to further analysis (Figure 2).
Data Analysis
The concentrations of dopamine, GABA, and glutamate were calculated considering the area under the curve of the chromatogram provided by Chromgraph software. No-net flux microdialysis data were analyzed as described by (Chefer et al., 2005, 2006). The amount of dopamine gained or lost from the probe during the no-net flux microdialysis (Cin − Cout) was calculated for each animal at each dopamine perfusion concentration (Cin: 0.0, 5.0, 10.0, 20.0, and 40.0 nM). The net change in dopamine (Cin − Cout) was plotted against Cin and subjected to linear regression (Figure 3a). The point at which no dopamine was gained or lost (Cin − Cout = 0) represents an estimate of dopamine Cext. The slope of the linear regression line represents the Ed, an indirect measure of dopamine transporter (DAT) activity. Basal dialysate dopamine levels were calculated for each animal as the average of the 3 basal samples (Cin = 0).
Each dopamine neuron record was analyzed using Neuroexplorer software, and the following parameters were determined: (1) the number of spontaneously active dopamine neurons per track, (2) average firing rate (number of spikes/time of the record), and (3) percentage of spikes in bursts (number of spikes in burst/total of spikes in record × 100). Statistical analyses were performed using Prism 5.0 GraphPad software. Data points outside of the 95% confidence interval were treated as outliers and could be excluded from the data analysis, although none were excluded in these studies. Normality was checked using the Kolmogorov–Smirnov test. Resultant data were analyzed by 2-way ANOVA, Sidak post-test, and 2-tailed unpaired t test when appropriate. All data are reported as mean ± SEM.
RESULTS
Effects of Repeated Exposure to WIN 55212-2 in Adolescence on DLS Dopamine Dynamics of Adult Rats
Adolescent exposure to WIN 55212-2 significantly increased basal dopamine dialysate in DLS of adult rats (Figure 3b; vehicle group: 0.55 ± 0.11 nM, n = 6 vs WIN group: 1.00 ± 0.12 nM, n = 6; P = .018, unpaired t test). The adolescent exposure to WIN did not modify dopamine Ed compared with the vehicle group (Figure 3c; vehicle group: 0.38 ± 0.07, n = 6 vs WIN group: 0.42 ± 0.06, n = 6; P = .51, unpaired t test). Consequently, dopamine Cext was higher in adult rats treated with WIN 55212-2 during adolescence compared with treated rats with vehicle (Figure 3d; vehicle group: 1.51 ± 0.13 nM, n = 6 vs WIN group: 2.42 ± 0.35 nM, n = 6; P = .033, unpaired t test).
Effects of Repeated Exposure to WIN 55212-2 During Adolescence on SNc Dopamine Neuron Activity
The adolescent administration of WIN 55212-2 increased significantly the spontaneous activity of dopamine neurons of the SNc (Figure 4b; vehicle group: 0.86 ± 0.09 cells/track, n = 7 vs WIN group: 1.29 ± 0.17 cells/track, n = 7; P = .046, unpaired t test). Differences in firing rate were not observed between groups treated with WIN 55212-2 and vehicle (Figure 4c; vehicle group: 3.10 ± 0.42 Hz, n = 36 neurons vs WIN group: 3.54 ± 0.51 Hz, n = 54 neurons; P = .60, unpaired t test). Adolescent exposure of WIN 55212-2 did not modify the percentage of spikes per burst compared with the vehicle group (Figure 4d; vehicle group: 17.55 ± 4.00%, n = 36 vs WIN group: 17.29 ± 3.43%, n = 54; P = .96, unpaired t test).
Effects of Repeated Exposure to WIN 55212-2 During Adolescence on Dialysate Levels of GABA and Glutamate in SNc of Adult Rats
Repeated exposure of WIN 55212-2 during adolescence decreased significantly basal GABA dialysate in SNc of adult rats (Figure 5a; vehicle group: 0.071 ± 0.006 µM, n = 9 vs WIN group: 0.051 ± 0.005 µM, n = 9; P = .03, unpaired t test). Adolescent exposure of WIN 55212-2 did not modify basal glutamate dialysate levels compared with the vehicle group (Figure 5b; vehicle group: 0.65 ± 0.10 µM, n = 9 vs WIN group: 0.57 ± 0.08 µM, n = 9; P = .54, unpaired t test).
Role of VP GABAergic Transmission on Dopamine Neuron Activity in SNc of Adult Rats After Adolescent Exposure to WIN 55212-2
A 2-way ANOVA, with adolescent treatment and VP perfusion as 2 independent factors, showed no significant effect of WIN 55212-2 treatment during adolescence (F1,27 = 0.997; P = .327) and bicuculline perfusion into the VP (F1,27 = 1.447; P = .235) on dopamine population activity. The interaction between these variables was significant (F 1,27 = 6.535; P = .017) (Figure 6a). Reproducing the results observed in Figure 4b, adolescent exposure to WIN 55212-2 increased significantly population activity of dopamine neurons in adult rats after a buffer perfusion into the VP (Figure 6a; buffer/vehicle group: 0.905 ± 0.11 cells/track, n = 7 vs buffer/WIN group: 1.39 ± 0.15 cells/track, n = 7; P = .046, Sidak post-test). As observed in VTA (Floresco et al., 2003), changes in dopamine neuron population activity were not observed in adult rats treated with vehicle after bicuculline perfusion (Figure 6a; buffer/vehicle group: 0.905 ± 0.11 cells/track, n = 7 vs bicuculline/vehicle group: 1.09 ± 0.16 cells/track, n = 9; P = .57, according to Sidak post-test). Perfusion of bicuculline into the VP reversed the increase in dopamine neuron population activity induced by adolescent exposure to WIN 55212-2 (Figure 6a; bicuculline/vehicle group: 1.09 ± 0.16 cells/track, n = 9 vs bicuculline/WIN group: 0.88 ± 0.10 cells/track, n = 8; P = .45, Sidak post-test; buffer/WIN group: 1.39 ± 0.15 cells/track, n = 7 vs bicuculline/WIN group: 0.88 ± 0.10 cells/track, n = 8; P = .028, Sidak post-test).
In addition, a 2-way ANOVA showed no significant effect of adolescent treatment of WIN55212-2 (F1187 = 1.615; P = .205) and bicuculline perfusion into the VP (F1187 = 3.570; P = .06) on dopamine neuron firing rate. The interaction was considered not significant (F 1187 = 0.065; P = .799) (Figure 6b). Also, a nonsignificant effect of WIN 55212-2 treatment during adolescence (F1187 = 1.79; P = .183) and bicuculline perfusion into the VP (F1187 = 0.21; P = .650) was observed on dopamine neuron burst firing pattern. The interaction was considered not significant (F 1187 = 0.001; P = .980) (Figure 6c).
Discussion
Cannabinoid use in the adolescent population has increased significantly in recent years across the world (UNODC, 2018). However, information regarding its long-term effects on brain activity is still sparse. Our study reveals that adolescent exposure to the cannabinoid agonist WIN 55212-2 renders the nigrostriatal pathway activated during adulthood. Electrophysiological and neurochemical approaches show an increase in the number of active dopamine neurons in the SNc accompanied by a significant increase in the dopamine Cext in DLS of adult rats. Interestingly, blocking GABAa receptors in the VP reverses the increase in dopamine neuron population activity in the SNc of adult rats treated with WIN 55212-2 during adolescence. The consequences of the VP-nigrostriatal circuit dysfunction induced by adolescent exposure to WIN 55212-2 on the limbic/cognitive/motor process during adulthood remains to be addressed.
The no-net flux experiments indicate that adolescent WIN 55212-2 is accompanied by an increase in DLS dopamine release without significant changes in Ed in the DLS of adult rats. An increase in dopamine release is consistent with previous evidence showing an increase of dopamine turnover in DLS of adult rats after repeated treatment with WIN 55212-2 (2 mg/kg) during early adolescence (PD 35–48) (Bortolato et al., 2014). Although an inhibitory effect of acute WIN 55212-2 on dopamine uptake has been observed in adolescent (Pérez-Valenzuela et al., 2019) and adult (Pandolfo et al., 2011) rats, no changes in dopamine Ed in DLS was evident in adult rats, suggesting that adolescent exposure to WIN 55212-2 is not accompanied by enduring modifications in DAT activity. In line with our observations, previous experiments of radioligand binding have shown that treatment with Δ 9-THC during early adolescence (PD 28–38) did not modify the binding of DAT in the dorsal striatum of adult male rats (Higuera-Matas et al., 2010). Collectively, the no-net flux experiments support the idea that the increase in dopamine Cext after adolescent exposure to WIN 55212-2 depends more on neuronal excitability than on pre-synaptic control involving dopamine uptake.
Studies have consistently reported that an increase in the activity of mesencephalic dopamine neurons produces an increase in dopamine efflux in the terminal regions (Floresco et al., 2003; Panin et al., 2012). Simultaneous microdialysis and extracellular recording experiments showed that an increase in firing rate and burst firing of SNc dopamine neurons increases basal dopamine dialysate in the DLS (Panin et al., 2012). In addition, evidence indicates that an increase in the proportion of spontaneous firing dopamine neurons produces an increase of basal dopamine dialysate (Floresco et al., 2003). Supporting the rise in dopamine Cext in DLS, in vivo single-unit recordings showed that adolescent exposure to WIN 55212-2 increases the population activity of dopamine neurons in the SNc without significant changes in firing rate or burst activity. A similar consequence of adolescent exposure to cannabinoids has been observed in VTA dopamine neurons (Gomes et al., 2015; Renard et al., 2017). Here, Gomes et al. (2015) showed that intermittent exposure to WIN 552112-2 during adolescence (PD 40–65) produces an increase in VTA DA neuron spontaneous activity (Gomes et al., 2015). Furthermore, Renard et al. (2017) showed that adolescent exposure to Δ 9-THC increases the firing rate, bursting activity, and population activity of VTA dopamine neurons (Renard et al., 2017). Together, our results indicate that the increase in dopamine Cext in DLS of adult rats after adolescent exposure to WIN 55212-2 depends on the facilitation of dopamine release supported by an increase in the number of spontaneously active dopamine neurons in the SNc.
It has been suggested that the number of spontaneously active dopamine neurons is regulated by GABA neurotransmission, while the burst pattern of dopamine neurons depends on glutamate neurotransmission (Floresco et al., 2003). Consistent with our recording data, microdialysis experiments showed a decrease in GABA extracellular concentration without changes in glutamate extracellular concentration in SNc of adult rats exposed to WIN 55212-2 during adolescence. Interestingly, a decrease in GABA neurotransmission in the adult medial prefrontal cortex has been observed after adolescent exposure to cannabinoid in males (Cass et al., 2014) and female (Zamberletti et al., 2014) rats. Accordingly, the decrease of GABA concentration in SNc along with attenuation of cortical inhibitory neurotransmission observed after adolescent exposure to cannabinoids (Cass et al., 2014) suggests a higher vulnerability in GABA neurotransmission during adolescence.
The extracellular GABA levels in the SNc are regulated by the endocannabinoid system. It has been described that the CB1 receptor is expressed in GABA inputs from dorsal striatum (Davis et al., 2018) and SNr (Freestone et al., 2014) onto SNc dopaminergic neurons. An increase in GABA postsynaptic currents is observed after the blocking of CB1 receptors, indicating an inhibitory tonic control of endocannabinoids on GABA levels (Freestone et al., 2014). Although the possible contribution of the striatal and SNr GABA inputs to our results cannot be ruled out, the repeated exposure to cannabinoids during the adolescence induces desensitization of CB1 receptors (Rubino et al., 2008), a result that would not account for the decrease in GABA extracellular levels observed in the SNc after adolescent exposure to WIN-55212-2.
As mentioned before, the SNc also receive monosynaptic inputs from the VP. Although cutting-edge neuroanatomical techniques have shown relatively low labeling between these regions, this does not imply a functional weakness (Watabe-Uchida et al., 2012). In fact, it has been shown that VP neurons modulate the population activity of dopamine neurons in the SNc (Bortz and Grace, 2018). Interestingly, CB1 receptors are expressed in terminal axons forming mainly inhibitory synapsis in the VP (Pickel et al., 2012), suggesting that repeated exposure to WIN 55212 could modify the VP control on dopamine neurons in the SNc. Therefore, single-unit recording experiments were carried out to assess if the activated state in the dopamine neurons of SNc after the adolescent cannabinoid exposure depends on VP control. Consistent with VTA observations (Floresco et al., 2003), bicuculline perfusion into the VP did not modify the population activity of SNc dopamine neurons in adult vehicle rats. Interestingly, blocking GABAa receptors in the VP reversed the increase in population activity in the SNc after adolescent WIN 55212-2 exposure. Our results suggest that the adolescence exposure to cannabinoid disinhibits SNc dopamine neurons, decreasing GABA inhibitory tone driven by VP. While acute exposure to WIN 55212-2 decreased the GABA extracellular levels in VP (Caillé and Parsons, 2006), the desensitization of CB1 signaling after repeated exposure to WIN 55212-2 (Rubino and Parolaro, 2008; Rubino et al., 2008) would increase the GABA tone in the VP, disinhibiting the nigrostriatal dopaminergic pathway.
To our knowledge, this study is the first evidence that shows the vulnerability and consequences of adolescent repeated exposure to cannabinoids in the nigrostriatal dopaminergic transmission of adult rats. Several questions remain unanswered at present. Further research is necessary to assess whether other GABA inputs contribute to the disinhibition of the nigrostriatal pathway after adolescent exposure to WIN 55212-2. Microdialysis experiments should be carried out to determine the effect of VP activation on the GABA and dopamine levels in the nigrostriatal pathway. Although previous evidence suggests an age-related vulnerability, it remains to be addressed whether these effects are observed during adult exposure to WIN 55212-2 and during long withdrawal after adolescent exposure to cannabinoids.
In summary, our results show that adolescent exposure to WIN 55212-2 increases the dopamine Cext in DLS accompanied by an increase in the population activity of dopamine neurons in SNc (Figure 7). In addition, this increase in dopamine neuron activity was reversed after blocking GABAa receptors in the VP. These pieces of evidence suggest that repeated activation of CB1 receptors during adolescence is accompanied by long-term consequences on the nigrostriatal dopaminergic pathway. As recently suggested, the activated state of dopaminergic nigrostriatal induced by adolescent cannabinoid exposure is a risk factor that could contribute to compulsive drug-seeking habit behavior during adulthood (Giuliano et al., 2019).
Acknowledgments
The authors acknowledge the services provided by UC CINBIOT Animal Facility funded by Program for Associative Research of the Chilean National Council for Science and Technology (PIA CONICYT) ECM-07 Dirección de Postgrado grant no. 391340281). This work was supported by “Fondo Nacional de Desarrollo Científico y Tecnológico” (FONDECYT #1141088 to J.A.F. and #1191152 to M.E.A.); “Proyecto de Mejoramiento Institucional, Pontificia Universidad Católica de Chile” (PMI PUC #1566); and “Comisión Nacional de Investigación Científica y Tecnológica,” Scholarship Program “DOCTORADO BECAS CHILE/2015” (CONICYT-PCHA #21150450 to E.P.V.).
Statement of Interest
The authors Enzo Pérez-Valenzuela, María Estela Andrés, and José Fuentealba Evans report no competing financial interests. Anthony A. Grace has received funds from Lundbeck, Pfizer, Otsuka, Lilly, Roche, Asubio, Abbott, Autofony, Janssen, Alkermes, Newron, and Takeda.