Animals and housing

Adolescent Sprague-Dawley rats were obtained at postnatal day (PND) 28 from Charles River Laboratories (Quebec, Canada). At arrival, rats were pair housed under controlled conditions (12 h light/dark cycle, constant temperature, and humidity) with free access to food and water. All procedures were performed in accordance with Governmental and Institutional guidelines for appropriate rat care and experimentation. The experimental protocols were approved by the Canadian Council on Animal Care and the Animal Care Committee at Western University, Ontario.

Adolescent THC administration

Rats treated with THC (Cayman Chemical) received twice daily injections of THC (2.5 mg/kg; Days 1–3; 5 mg/kg; Days 4–7; 10 mg/kg, Days 8–11). Control groups received the same injection schedule (volume adjusted per body weight) with vehicle (VEH). Increasing doses of THC were administered to counter the development of drug tolerance80. These doses of THC were chosen based on our and others previously published protocols6, 50, 81,82,83 and are known to produce long-term behavioral and molecular and neuronal effects in rats. Importantly, this escalating THC exposure protocol mimics a heavy use regiment of marijuana exposure, with the 2.5 mg/kg dose corresponding to approximately one cannabis cigarette, the 5 mg/kg dose corresponding to approximately two cannabis cigarettes, and the 10 mg/kg dose corresponding to approximately four cannabis cigarettes50. THC in ethanol was dissolved in cremophor and saline (1:1:18). Ethanol was then evaporated using nitrogen gas to remove it from the final THC solution. All injections were administered intraperitoneally (i.p). The adolescent exposure experiments began at PND 35. Experimental procedures were initiated following a 30 days drug-free period (at PND 75).

Protein Expression Analyses

At adulthood (PND75), rats received an overdose of sodium pentobarbital (240 mg/kg, i.p., EuthanylTM). Under deep anesthesia rats were decapitated and brains removed and frozen. Coronal sections (60 μm) containing the PFC were cut on a cryostat and slide mounted. Bilateral micro-punches of the mPFC, were obtained for protein isolation. The western blotting procedure was performed as described previously84. Primary antibody dilutions were as follows: α-tubulin (1:120 000; Sigma-Aldrich), GAD67 (1:1000; Cell Signaling Technology), GAD65 (1:200; Santa Cruz Biotechnology) and Parvalbumin (1:2000; Sigma-Aldrich). Species appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Thermo Scientific) were all used at a dilution of 1:20 000.

Neuronal Activity Recordings and Analysis

Extracellular single-unit electrophysiological recordings were performed in vivo in adult (>PND 75) VEH and THC pretreated rats during adolescence. The recordings were taken from either putative glutamatergic (GLUT) PFC neurons (cells/animals: VEH = 99/6, THC = 122/10) or dopaminergic (DA) VTA neurons (cells/animals: VEH = 45/24, THC = 40/20). Rats were anesthetized with urethane (1.4 g/kg, i.p., Sigma-Aldrich) and placed in a stereotaxic frame with body temperature maintained at 37 °C. A scalp incision was made and a hole was drilled in the skull overlaying the targeted structure at the following coordinates: mPFC: AP: +2.7 to +3.5 mm from bregma, L: ±0.8 to ±1 mm, DV: −2.5 to −4 mm from the dural surface; VTA: AP: −5.2 mm from bregma, L: ±0.8 to ±1 mm, DV: −6.5 to −9 mm from the dural surface. Recordings were taken with glass microelectrodes (average impedance of 6–10 MΩ) filled with 2 M sodium acetate solution containing 2% pontamine sky blue (Sigma-Aldrich). A bone screw was placed over the cerebellum and was connected with the return of the headstage and served as a reference electrode. Extracellular signals were amplified (×5000) using a MultiClamp 700B amplifier (Molecular Devices), digitized at 25 kHz and recorded on the computer using a Digidata 1440 A and pClamp software (Molecular Devices). The wideband signal of PFC recordings was fed to two channels of the digitizer and filtered to obtain single unit recordings (band pass between 0.3 and 3 kHz) and local field potentials (LFPs; low pass at 0.3 kHz). For the VTA electrophysiology only unit data were recorded.

Putative mPFC pyramidal cells were identified based on established criteria85: (1) firing frequency < 10 Hz, (2) waveform shape, and (3) action potential duration > 2.5 ms. Cells exhibiting 3 consecutive spikes with inter-spike intervals < 45 ms were classified as burst-firing cells85. The electrophysiological properties of spontaneously active pyramidal neurons were sampled in the mPFC by making vertical passes of the electrode through the pyramidal cell body region. These tracks were made in a predefined pattern, with each track separated by 200 µm. After an individual putative pyramidal neuron was isolated, its spontaneous activity was recorded for 5 min. Two parameters of activity were sampled, the basal firing rate and the bursting rate.

VTA DA neurons were identified according to well established electrophysiological features86: (1) action potential width > 2.5 ms, (2) spontaneous firing rate between 2–5 Hz, (3) a triphasic waveform consisting of a notch on the rising phase followed by a delayed after potential, and (4) a single irregular or burst firing pattern. For intra-mPFC microinfusions of VEH or Muscimol (MUS, 1 μg/0.5 μl) a 10 μl Hamilton syringe was slowly lowered into the mPFC using the same stereotaxic coordinates as described above. The firing frequencies of collective VTA putative DA neurons from adolescent THC pretreated rats before microinfusions of VEH or muscimol into the mPFC were averaged and normalized to the average firing frequency of collective VTA neurons from adolescent VEH pretreated rats. The response patterns of isolated VTA neurons to microinfusion of VEH or MUS into the mPFC were determined by comparing the neuronal frequency rates between the 5 min preinfusion vs. postinfusion recording epochs. We also analyzed the proportion of DA neuronal spikes fired in burst mode. The onset of a burst was defined as the occurrence of two consecutive spikes with an interspike interval of 80 ms86. The percentage of burst spikes was calculated by dividing the number of spikes occurring in bursts by the total number of spikes occurring in the same period of time. We sampled a total of n = 85 VTA DA neurons (Adolescent VEH pretreated-Intra mPFC VEH group; n = 15 cells in 7 rats; Adolescent VEH pretreated- Intra-mPFC MUS group; n = 30 cells in 17 rats; Adolescent THC pretreated- Intra mPFC VEH group; n = 14 cells in 7 rats; Adolescent THC pretreated- Intra mPFC MUS group; n = 26 cells in 13 rats).

LFP signals were analyzed using NeuroExplorer (Nex Technologies). First, the signals were decimated to 1 kHz, and lowpass filtered (IIR Butterworth filter at 170 Hz; filter order set to 3). Subsequently a spectrogram function was used to calculate the power of oscillations at frequencies between 0–100 Hz (window length 2 s; shift 0.5 s). One minute long recording epochs were used for estimating the average power spectrum distributions. Epochs were selected such as either the desynchronized (relatively small signal amplitude devoid of slow oscillations) or synchronized (relatively large signal amplitude with slow oscillations present) cortical state could be easily distinguished. Power values for a given frequency were averaged over time of the recording epoch and normalized so that the sum of all power spectrum values equals 1. The total power was calculated by adding all the power values at frequencies between 0–59 and 61–100 Hz. Power values at 60 ± 1 Hz were excluded from all the calculations. Gamma band was defined as frequency between 30–80 Hz and subcategorized into low gamma (30–59 Hz) and high gamma (61–80 Hz). Statistics are based on 98 recordings of desynchronized state (VEH = 37, THC = 61) and 174 recordings of synchronized state (VEH = 107, THC = 67). Every recording was taken at different electrode location throughout mPFC.

For histological analyses the recording electrodes positions were marked with an iontophoretic deposit of pontamine sky blue dye (−20 mA, continuous current for 15 min). Histological analysis was performed as described previously47. No mPFC cells were recorded outside the anatomical boundaries of the mPFC as defined by Paxinos and Watson (2007). One recorded rat was excluded from electrophysiological data analysis because its cells were recorded outside the anatomical boundaries of the VTA as defined by Paxinos and Watson (2007).

Surgical Procedures

At adulthood (PND) 75, rats were anesthetized with an intraperitoneal injection of ketamine (80 mg/mL, Vetoquinol)-xylazine (6 mg/kg, Bayer) mixture. To minimize pain and inflammation, meloxicam (1 mg/kg; s.c., Boehringer Ingelheim) was administered before and after surgeries. Rats were positioned in a Kopf stereotaxic device for cannulae implantation. Stainless steel guide cannulae (22-gauge) were implanted bilaterally into the mPFC with the coordinates (15° angle, in mm from bregma): AP: +2.9 L: ±1.9 mm: DV: −3 mm from the dural surface. The guide cannulae were secured into position by jeweler’s screws and dental acrylic cement. Rats were singly housed post-surgery and behavioral tests were initiated after one week of recovery.

Intra-mPFC Microinfusions

Intra-mPFC microinfusions of either vehicle (VEH, NaCl 0.9%) or the selective GABA A receptor agonist, muscimol (500 ng/0.5 µl, Sigma-Aldrich, diluted in NaCl 0.9%) were performed immediately before behavioral experiments. A total volume of 0.5 μl per hemisphere was delivered via a 28 gauge injector over a period of 1 min. Microinjectors were left in place for an additional 1 min following infusions to ensure adequate diffusion from the tip.

Behavioral Testing

Object recognition

Rats were tested using the object recognition task as described previously87. This task evaluates the ability of the rat to discriminate between the familiarity of previously encountered objects; normal rats typically spend more time exploring a novel object than a familiar object. The test sessions consisted of two 3-min trials. During the first trial (T1 acquisition trial), each rat was placed in the center of an arena containing two identical objects placed in the far corners 15 cm from the side wall. After a delay of 60 min during which the rat was returned to its cage, and both objects were replaced (one by an identical copy, the other by a novel object in the same location), the rat was returned to the arena for the second trial (T2 test trial). Between rats, both the role (familiar or novel object) and the relative position of the two objects were randomly counterbalanced. Object exploration was considered when the head of the rat was facing the object or the rat was touching or sniffing the object. Times spent in exploration were videotaped with a video-tracking system (ANY-maze; Stoelting) and analyzed by an experimenter blind to the treatment conditions. Exploration times were recorded and used to calculate object recognition index [time spent with novel object/total time exploring both objects] *100. The final number of rats in each group was as follows: Adolescent VEH-Intra-mPFC VEH (VEH/VEH) group, n = 10; Adolescent VEH-Intra-mPFC MUS (VEH/MUS) group, n = 9; Adolescent THC-Intra-mPFC VEH (THC/VEH) group, n = 9; Adolescent THC-Intra-mPFC MUS (THC/MUS) group, n = 10.

Social motivation and social cognition

Rats were tested using a social interaction procedure as described previously6. Briefly, this task evaluates 2 distinguishable aspects of social behavior: (1) social affiliation/motivation and (2) social recognition memory. Rats were habituated to the test arena for 13 min, 24 h before testing. Testing consisted of 2 successive 8-min phases. During the first phase, we analyzed social motivation, that is, the propensity to spend time with an unfamiliar male rat (stranger rat) enclosed in a small wire cage compared with time spent with an identical but empty cage. During the second phase, occurring just after the first one, we analyzed social recognition, that is, the propensity to spend time with a novel unfamiliar rat (novel stranger) rather than with the familiar stranger rat (encountered during the first phase). The locations of stranger vs. novel rats in the left vs. right side chambers were counterbalanced between trials. Times spent in exploration were videotaped with a video-tracking system (ANY-maze; Stoelting) and analyzed by an experimenter blind to the treatment conditions. After each test, chambers and cages were cleaned with 50% ethanol to avoid olfactory cue bias. Exploration times were recorded and used to calculate a social motivation or cognition index [time spent with stranger (or novel stranger)/total time exploring both rats] *100. The final number of rats in each group was as follows: Adolescent VEH-Intra-mPFC VEH (VEH/VEH) group, n = 12; Adolescent VEH-Intra-mPFC MUS (VEH/MUS) group, n = 11; Adolescent THC-Intra-mPFC VEH (THC/VEH) group, n = 12; Adolescent THC-Intra-mPFC MUS (THC/MUS) group, n = 10.

Light dark box

This test is based upon a rat’s natural aversion to bright environments and attributes greater time spent in an illuminated environment as reflecting lower anxiety levels. The test was performed as previously described6. At the start of the experiment, a rat was placed in the center of the lighted box with its head facing the wall opposite the door and was allowed to freely explore both compartments for a period of 8 min. A zone entry was considered to have begun when the rat placed all 4 paws in that zone. Experiments were videotaped with a video-tracking system (ANY-maze;Stoelting) and analyzed by an experimenter blind to treatment conditions. Behaviors analyzed included latency time to leave the dark box and enter the light box (latency to second transition), which is thought to be the most reliable indicator of anxiety-like behavior and is sensitive to both anxiogenic and anxiolytic treatments88. The final number of rats in each group was as follows: Adolescent VEH-Intra-mPFC VEH (VEH/VEH) group, n = 9; Adolescent VEH-Intra-mPFC MUS (VEH/MUS) group, n = 12; Adolescent THC-Intra-mPFC VEH (THC/VEH) group, n = 12; Adolescent THC-Intra-mPFC MUS (THC/MUS) group, n = 10.

Open field test

Rats were placed in an automated open field activity chamber (San Diego Instruments, San Diego, CA, USA) for 60 min. Total distance travelled and vertical counts were recorded and analyzed. The final number of rats in each group was as follows: Adolescent VEH-Intra-mPFC VEH (VEH/VEH) group, n = 11; Adolescent VEH-Intra-mPFC MUS (VEH/MUS) group, n = 12; Adolescent THC-Intra-mPFC VEH (THC/VEH) group, n = 12; Adolescent THC-Intra-mPFC MUS (THC/MUS) group, n = 9.

Statistical analysis

The data were analyzed using t-tests, one or two-way ANOVA. Post hoc analyses were calculated using Fisher’s LSD. Densitometry values for Western blots were acquired with Kodak digital analysis software and analyzed with t-tests.

Data availability

The authors declare that the data supporting the findings of this study are available from the corresponding author on reasonable request.