Lysergic acid diethylamide (LSD) promotes social behavior through mTORC1 in the excitatory neurotransmission

Significance Social behavior (SB) is a fundamental hallmark of human interaction. Repeated administration of low doses of the 5-HT2A agonist lysergic acid diethylamide (LSD) in mice enhances SB by potentiating 5-HT2A and AMPA receptor neurotransmission in the mPFC via an increasing phosphorylation of the mTORC1, a protein involved in the modulation of SB. Moreover, the inactivation of mPFC glutamate neurotransmission impairs SB and nullifies the prosocial effects of LSD. Finally, LSD requires the integrity of mTORC1 in excitatory glutamatergic, but not in inhibitory neurons, to produce prosocial effects. This study unveils a mechanism contributing to the role of 5-HT2A agonism in the modulation of SB.


Single and repeated LSD treatment
For single administration experiments, mice were injected with vehicle (veh) or LSD (30 μg/kg) and were tested 30 min and 24 h after the injection. This relatively low dose for an animal study was chosen because it decreases the 5-HT firing activity of the Dorsal Raphe Nucleus (DRN) without affecting the dopaminergic neurons of the Ventral Tegmental Area (VTA) (1). This low dose does not produce stereotypies, does not affect locomotion, nor does it stimulate the dopaminergic system as LSD does when administered at high doses (2). For repeated administration, mice and rats received LSD (30 μg/kg/day, i.p., for 7 days) and were tested 24 h after the last injections for behavioral, electrophysiological and biomolecular evaluation.

In Vivo Electrophysiology
Preparation for recording procedures.
Firstly, the mice were anesthetized with urethane (1.4 g/kg, intraperitoneal), then mounted on a stereotaxic frame (David Kopf Instruments, Tujunga, California) with the skull positioned horizontally. Anesthesia was confirmed by the absence of nociceptive reflex reaction to a tail or paw pinch and lack of eye blink response to pressure. Body temperature was maintained at 37 ± 0.5 C throughout the experiment using a thermistorcontrolled heating pad (Seabrook Medical Instrument, Inc., Seabrook, NH, USA). Recordings were carried out using microiontophoresis multi-barreled (Harvard/Applied Scientific Instrumentation, OR, USA) glass micropipettes pulled on a Narashige (Tokyo, Japan) PE-2 pipette puller. The micropipettes were preloaded with fiberglass strands to promote capillary filling with 2% Pontamine Sky Blue solution in 2M NaCl, and their tips were broken down to diameters of 1-3 mm for single-barreled and 10-15 mm for multi-barreled recordings. The impedances ranged from 2 to 6M. The stereotaxic brain coordinate system by Paxinos and Franklin (6) was used in all electrophysiological experiments. Using a hydraulic micropositioner (model 650; David Kopf Instruments, Tujunga, California), the electrode was advanced slowly into the brain structure at approximately 0.15 mm/min to minimize the probability of missing slow-spiking neurons. To maximize sampling without introducing considerable tissue damage, three to five electrode descents were performed. Single-unit activity was recorded as discriminated action potentials amplified by a Tennelec (Oakridge, TN) TB3 MDA3 amplifier, post-amplified and filtered by a Realistic 10 band frequency equalizer, digitized by a CED1401 interface system (Cambridge Electronic Design, Cambridge, UK), processed online, and analyzed off-line using Spike2 software version 5.20 for Windows PC (Microsoft, Seattle, WA).
A Npi electronic Gmbh Microiontophoretic System (Tamm, Germany) was used for local (iontophoretic) drug applications. The spontaneous single-spike activity of neurons was recorded for at least 2 min; the first 30 s immediately after detecting the neuron was not considered to eliminate mechanical artifacts due to electrode displacement. For experiments requiring acute drug injection, a catheter was inserted intraperitoneally prior to electrophysiological recording to facilitate intraperitoneal administration. Drug response was considered inhibitory if the drug decreased the basal firing rate of a neuron by at least 10%.
At the end of each recording session, the recording site was marked by iontophoretic ejection (1-10 mA, negative current for 10min) of Pontamine Sky Blue for later histological verification of recording sites. All recordings were carried out between 1400 and 2200 hours. A first cohort of mice (n=4) underwent in vivo electrophysiology combined with optogenetic photo-inhibition in the mPFC. Another group of mice treated with a single dose of LSD (30 g/kg, i.p.) underwent single unit extracellular recordings of pyramidal neurons in the mPFC. Then mice treated with repeated LSD (30 g/kg/day, i.p., for 7 days) underwent single unit extracellular recordings of pyramidal neurons in the mPFC as reported in Supplementary.
Raptor f/f :Camk2-Cre and their littermates treated with LSD (30 g/kg/day, i.p., for 7 days) or veh underwent in vivo electrophysiological recordings in the mPFC. A total of 33 mice were used for these experiments

Extracellular recordings and microiontophoresis from the medial prefrontal cortex
This procedure was performed according to our protocols (7,8). The multi-barreled micropipette was lowered into the prelimbic (PL) and infralimbic (IL) region of the ventromedial prefrontal cortex (mPFC) (1.5-2.0 mm anterior to bregma; 2.5-3.5 mm ventral to the dura mater; 0.25 mm from the midline, within layer 5 of the cortex). The side barrels had impedances ranging from 50 to 150 M and contained NMDA, 8-OH-DPAT, DOI, or quisqualic acid, and a NaCl solution (2 M) for automatic current balancing. As most of the pyramidal neurons are not spontaneously active under anesthesia, prolonged low-current NMDA ejections (-5nA) were introduced to activate them within their physiological firing rates (0.5-10 Hz in the mPFC) (7,9,10). It has been shown that there is no response difference between pharmacologically induced, and spontaneously firing pyramidal neurons (9). Neurons were also identified based on their steady response to standard short pulses of NMDA and by large amplitude, long duration, and single-action potential patterns alternating with complex spike discharges (7,8,11). Employing increasing currents (-20 to -50 nA, 30 s currents), NMDA was also used to assess the sensitivity of this receptor. DOI was used to assess the sensitivity of 5-HT2A receptors and was ejected as an anion (-20 to -50 nA, 30 s currents) and retained with a current of 20 nA. Quisqualate was used to assess the sensitivity of α-amino-3-hydroxy-5-methyl-4isoxazole propionate (AMPA) receptor and was ejected as an anion (-20 to -50 nA, 30 s currents) and retained with a current of 20 nA. 8-OH-DPAT was used to assess the sensitivity of 5-HT1A receptors and was ejected as a cation (+20, +30 and +50 nA in the mPFC, 30 s currents) and retained with a current of -20nA. Pyramidal activity was identified by large amplitudes (0.5-1.2 mV), long durations (0.8-1.2 ms), and as single action potentials alternating with complex spike discharges. Pyramidal neural response to systemic or microiontophoretic drug application was expressed as percentage increase/decrease from predrug microionthophoretic application baseline (0 nA current). For bursting analysis, cells exhibiting 3 consecutive spikes with inter-spike intervals < 45 ms were classified as burst-firing cells (12).

Direct Social Interaction (mice)
This test was performed according to our standardized protocol (13). Test mice were placed in a clean cage and given 10 min to habituate. Immediately after habituation, a novel age-and sex-matched conspecific stranger mouse was introduced into the cage and the mice were able to freely engage in social interaction for 10 min. The interaction time, defined by the following behaviors: nose-to-anogenital sniffing, nose-tonose sniffing, and social grooming, was manually scored.

Direct Social Interaction (rats)
This test was adapted from our protocol (14). After receiving repeated administration with veh or LSD (30 g/kg/day, for 7 days), Sprague Dawley rats were placed in an open field arena (80 x 80 x1 5 cm) and given 10 min to habituate. Immediately after habituation, a novel age-and sex-matched conspecific stranger rat was introduced into the cage and the rats were able to freely engage in social interaction for 10 min. The interaction time, defined by the following behaviors: nose-to-anogenital sniffing, nose-to-nose sniffing, and social grooming, was manually scored. The test was performed 24 hours later the last injection of LSD or veh.

Three-Chamber Test
This test was performed according to our standardized protocol (13,15). A three-chamber arena with openings between the chambers was used to assess sociability and preference for social novelty. Test mice were placed in the middle chamber and allowed to freely explore the empty three-chamber arena for 10 min. Immediately after habituation, an unfamiliar mouse (stranger 1, male C57BL/6J, age matched) was introduced into 1 of the 2 side chambers, enclosed in a wire cage, thus allowing only the test mouse to initiate social interaction. An identical empty wire cage was placed in the other side chamber. With this setup, the test mouse was again placed in the middle chamber and allowed to explore the three-chamber arena for 10 min. At the end of the 10-min sociability test, a new unfamiliar mouse (stranger 2, male C57BL/6J, age matched) was placed in the previously unoccupied wire cage. The test mouse was observed for an additional 10 min to assess social novelty (as explained below). The location of the empty wire cage was alternated between side chambers for different test mice to prevent chamber biases. Stranger 1 (S1) and 2 (S2) mice were always taken from separate home cages and counterbalanced for each side of the chamber apparatus and stranger cage. The time spent interacting with S1, S2, or the empty cage, was manually scored. The interaction time was determined by measuring the duration of the head/body contacts or climbing of the subject mouse upon either the empty cage or the cage containing the stranger mouse In order to ensure a comparisons across treatments, strangers and genotype groups, and also to reduce the impact of variable exploration times between mice, we used a "sociability index" for each mouse, calculated as: 100 × (S1 interaction time− empty cage interaction time)/(S1 interaction time + empty cage interaction time) and a "social novelty index" for each mouse, calculated as: 100 × (S2 interaction time− S1 interaction time)/(S2 interaction time + S1 interaction time) (16). Number of contacts upon the empty cage or the cage containing the S1/S2 mouse were also included, as well as the percentage of preference for the empty cage (empty cage interaction time/ (empty cage interaction time+ S1 interaction time) x 100) during the sociability phase. Mice were excluded from further analysis if they either failed to explore the empty cage or the mouse S1/S2 chambers, or if they spent more than 75% of the allotted time in the center chamber, not exploring either chambers containing S1, S2, or the empty cage. For these reasons, 19 mice out of 190 undergoing the TCT were excluded (171 in total). All stranger mice were purchased from Charles River Laboratories (Sherbrooke, QC)

Open field activity testing
According to our protocols (7), mice were individually placed at the center of a white-painted open field arena (40 x 40 x 15 cm) and left to explore the whole arena for 20 min. The experiment took place under standard room lighting (350 lx); a white lamp (100 W) was suspended 2 m above the arena. Anxiety-like thigmotactic ('wall-following') behavior was measured by the frequency and total duration of central zone (30 x 30 cm) visits. Other ethological measures analyzed included grooming, rearing and locomotor activity.

Novelty-suppressed feeding test
According to our protocols (4), this procedure was used to measure novelty-induced anxiety-like behavior (neo-hypophagia, the inhibition of feeding upon exposure to an anxiogenic novel environment). This test has been widely used to validate the acute effects of putative anxiolytics. The mice were food-deprived for The cut-off time was 600s (Feeding latency was also observed in the home cage containing 3 pellets spread on the floor to exclude the possibilities that mice were not hungry; the session was terminated immediately after the mice initiated feeding).

Forced swim test
According to our protocol (17), mice were individually placed into Plexiglas cylindrical bins (20 cm diameter, 50 cm high) filled with water (25-27 °C) to a depth of 20 cm. This depth did not allow the tail or the hind paws to touch the bottom of the bin. Mice were allowed to swim for 6 min. Infrared light-sensitive CCD cameras allowed for the capture and storage of images with the videotrack system (View Point Life Science, Montreal (QC), Canada)). After recording, mice were rescued using a plastic grid and placed in a cage near a heat lamp to dry. The behavioral tracking system was calibrated so that a mouse was considered immobile when making only minimal movements necessary to keep its head above the water. The total duration of activity was determined during the last 4 min.

Sucrose Preference Test
This test was performed employing our protocols (17). Mice were individually housed 3 days before the beginning of the test. They were then trained for 3 days to consume water from two bottles. During these 3 days, the two bottles containing water were replaced for 1 h a day with two bottles filled with a 2% (w/v) sucrose solution. Next, mice were subjected to a 48h procedure during which they were allowed to discriminate and select between 2 drinking bottles, one containing water and the other the sucrose solution.
To avoid conditioned place preference learning, the bottles were placed on the home cage for 48 h (starting at the beginning of the light phase, 7:00 A.M.) and their positions were interchanged in the mid-point of each light (1:00 P.M) and dark (1:00 A.M.) cycle over these 2 days. The sucrose preference (%) was determined as follows: sucrose solution intake (g)/total fluid intake (g) × 100. were fixed to the skull using dental cement and a pair of skull screws.

Cannula implantation for intra-mPFC infusion
The intra-mPFC antagonist infusions experiments followed the same procedure as above. Mice were implanted with internal cannulae (Plastics One, HRS Scientific, Canada) above the mPFC extending 2 mm below the cannulae guides as explained in Supplemental. Microinfusions of aCSF, NBQX or MDL occurred at a rate of 0.1 ml/min over 2 minutes, once per day, for 7 days, 10 min before the systemic injection of LSD or veh (saline). To maximize diffusion, the internal cannulae were kept in place for an additional 2 minutes after the infusion. 24 hours after the last infusion, locomotor impairment was assessed in the OFT.

Optogenetic manipulations
Photo-inhibition was performed employing a modified protocol (18). For in vivo electrophysiology recordings of mPFC pyramidal neurons, C57BL/6J mice were anesthetized and placed on a stereotaxic frame.  (19). For behavioral experiments, another cohort of mice treated with LSD or veh for 7 days were tested for DSI. In this case, each animal coupled to a fiberoptic patch cord was connected to a fiberoptic rotary joint (Doric, Quebec). The rotary joint was connected via the patch cord to the dual LD fiber light source for optogenetic inhibition. Mice underwent habituation to the patch cord in their home cage 5 min per day, for two days. The last habituation was performed 24 hours before the experiment. The day of the test, both the control group and LSD-treated animals (last LSD dose 24 hours before the test) were connected to the laser and, after 10 min of habituation, they underwent the DSI and received the green light (intensity, 10 mW; 530-nm laser) for 10 min (continuous light). The light was delivered immediately after the introduction of the intruder mouse and it lasted for the duration of the test. The same cohort of mice underwent the DSI again 24 hours later (using different intruder mice coming from different cages) with the light turned OFF. This inhibition protocol has been demonstrated not to bleach or injure brain tissue (20,21).

Histological verification of viral expression
At the end of each optogenetic experiment, animals were anesthetized with a ketamine/xylazine (120 mg/kg/10 mg/kg) cocktail and transcardially perfused with 4% paraformaldehyde in phosphate-buffered saline. Brains were removed and fixed in 4% paraformaldehyde for 6-12 h; they were then placed in 30% sucrose for 48-72 h before freezing. For viral expression verification, brains were sectioned into 25-μm slices (Leica VT1000s) and mounted with MOWIOL plus DAPI solution (Sigma-Aldrich, Oakville, Ontario, Canada). mPFC sections (PL and IL) were imaged on a confocal microscope (LSM710, Zeiss, McGill University Cell Imaging and Analysis Network).