Effect of delta-9-tetrahydrocannabinol on behavioral despair and on pre- and postsynaptic serotonergic transmission

Progress in Neuro-Psychopharmacology & Biological Psychiatry 38 (2012) 88–96

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Effect of delta-9-tetrahydrocannabinol on behavioral despair and on pre- and postsynaptic serotonergic transmission Francis Rodriguez Bambico, Patrick R. Hattan, Jean-Philippe Garant, Gabriella Gobbi ⁎ Neurobiological Psychiatry Unit, Dept. Psychiatry, McGill University, Montréal, Québec, Canada

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Article history: Received 9 November 2011 Received in revised form 2 February 2012 Accepted 13 February 2012 Available online 22 February 2012 Keywords: Behavioral despair Cannabinoids CB1 receptor Delta-9-THC Serotonin

a b s t r a c t Preclinical and clinical studies suggest that direct and indirect cannabinoid agonists, including enhancers of endocannabinoids, engender stress-relieving, anxiolytic and antidepressant effects, mediated by central CB1 receptors (CB1Rs). The effect of the main pharmacologically active principle in cannabis, (−)-trans-Δ9tetrahydrocannabinol (delta-9-THC), on depressive behavior and on the serotonin (5-HT) system, which is implicated in the mechanism of action of antidepressants, has not been extensively clarified. Here, we showed that repeated (5 days), but not single (acute) intraperitoneal (ip) treatment with delta-9-THC (1 mg/kg) exerts antidepressant-like properties in the rat forced swim test (FST). This effect was CB1R-dependent because it was blocked by the CB1R antagonist rimonabant (1 mg/kg, ip). Using in vivo electrophysiology, we demonstrated that delta-9-THC modulated dorsal raphe (DR) 5-HT neuronal activity through a CB1R-dependent mechanism. Acute intravenous delta-9-THC administration (0.1–1.5 mg/kg) elicited a complex response profile, producing excitatory, inhibitory and inert responses of 5-HT neurons. Only excitatory responses were blocked by rimonabant. Finally, repeated but not single delta-9-THC administration (1 mg/ kg, ip) enhanced tonic 5-HT1A receptor activity in the hippocampus, a postsynaptic event commonly elicited by standard antidepressants. These results suggest that delta-9-THC, like other CB1R agonists and endocannabinoid enhancers, may possess antidepressant properties at low doses, and could modulate 5-HT transmission in the DR and hippocampus as standard antidepressants such as selective serotonin reuptake inhibitors. © 2012 Elsevier Inc. All rights reserved.

1. Introduction Major depression is one of the most prevalent and debilitating psychiatric afflictions. It is characterized by pervasive and recurrent episodes of low mood and motivation, despair and anhedonia. Despite the availability of several classes of antidepressants, more than a third of patients are either unremitting or relapsing (Bambico and Gobbi, 2008; Zisook et al., 2008). Antidepressants that are known to act on the brain's monoamine systems are limited by a delayed onset of therapeutic action. Moreover, the pathophysiology of depression is far from being fully elucidated. Multiple levels of dysfunction have been proposed, including impaired synaptic transmission of the monoamine 5-hydroxytryptamine (5-HT or serotonin) (Bambico et al., 2009a), which is produced by the midbrain raphe 5-HT neurons (Dahlström and Fuxe, 1964; Descarries et al., 1982), and known to regulate emotional, vegetative and neuroendocrine Abbreviations: 5-HT, 5-hydroxytryptamine, serotonin; CB1R, cannabinoid CB1 receptor; Delta-9-THC, (-)-trans-delta(9)-tetrahydrocannabinol; DR, dorsal raphe; FAAH, fatty acid amide hydrolase; MAGL, monoacylglycerol lipase; FST, forced swim test; OFT, open field test; WAY100, 635, N-[2-[4-(2-methoxyphenyl-1-piperazinyl] ethyl]-N-(2-pyridyl)cyclohexanecarboxamide. ⁎ Corresponding author at: 1033 Pine Avenue West, Neurobiological Psychiatry Unit, Department of Psychiatry Research and Training Building, McGill University, Montréal, Québec, Canada H3A 1A1. Tel.: + 1 514 398 1290; fax: + 1 514 398 4866. E-mail address: [email protected] (G. Gobbi). 0278-5846/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2012.02.006

functions (Holmes, 2008). Not surprisingly, drugs that augment 5-HT transmission, such as selective serotonin reuptake inhibitors (SSRIs), are most preferred first-line treatments (Vaswani et al., 2003). Their chronic application facilitates exocytosis of 5-HT and/or increases 5-HT synaptic availability in corticolimbic regions extensively innervated by the raphe. Many of these forebrain structures, including the prefrontal cortex, hippocampus and amygdala are implicated in mood regulation and stress adaptation (Holmes, 2008). On the other hand, chronic stress that is known to be a risk factor for depression impairs presynaptic and postsynaptic 5-HT transmission (Bambico et al., 2009b). Also, acute or repeated 5HT or tryptophan depletion precipitates anxiety/depression-like behaviors in animals (Blokland et al., 2002) and mood-lowering effects in humans (for review, Young and Leyton, 2002), exacerbates anxiogenic responses in human subjects (Miller et al., 2000), and triggers relapse in depressive patients (for review, Van der Does, 2001). The role of the 5-HT1A receptor in the antidepressant response is well established. The therapeutic onset of many antidepressants has been attributed to gradual neuroplastic adaptations of these receptors, such as to the desensitization of presynaptic dorsal raphe (DR) 5-HT1A auto-inhibitory receptors and to the enhancement of the tonic activity of postsynaptic hippocampal 5-HT1A receptors (for review, Bambico and Gobbi, 2008; Bambico et al., 2009a). Such has been hypothesized to result from the progressive augmentation in 5-HT activity, and that could be linked to the neurogenic effects on hippocampal cells,

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observed after chronic antidepressant treatment. In addition to 5-HTspecific modes of action, agents that enhance the transmission of norepinephrine (NE) have also been shown effective in alleviating depressive symptoms, likely due to the interplay of 5-HT and NE neurotransmission in correcting or in compensating for deficiencies produced by the disease (for review, Bambico and Gobbi, 2008). The main pharmacologically active cannabinoid principle in cannabis, (−)-trans-Δ9-tetrahydrocannabinal (delta-9-THC), likely mediates most of its psychoactive and mood-related effects (Huestis et al., 2001). This is known to be achieved by activation of cannabinoid CB1 receptors (CB1R), one of two well-characterized, and the most abundant G-protein coupled receptor in the mammalian brain. Although heavy or high-dose cannabis use has been associated with escalated risks for mood disorders, anxiety, psychosis and cognitive impairment, especially among teen-agers, its continued use for self-medicating depressive symptoms suggests possible therapeutic benefits in primary and secondary depression (for review, Bambico and Gobbi, 2008). Recently, the lipid endocannabinoid molecules Narachidonoylethanolamide (anandamide) and 2-arachidonoylglycerol (2-AG)- along with their native receptor, the CB1R; and their catabolic enzymes, fatty acid amide hydrolase (FAAH) mainly for anandamide, and monoacylglycerol lipase (MAGL) mainly for 2-AG- have been found to be present in 5-HT neurons in the DR (Egertova et al., 1998; Häring et al., 2007; Moldrich and Wenger, 2000). In addition to their localization in monoaminergic neurons, these endocannabinoid elements are also expressed in excitatory (glutamatergic) and inhibitory (GABAergic) neurons throughout the cerebral cortex (for review, Esteban and Garcia-Sevilla, 2011). CB1R signaling in the DR 5-HT system has been shown to modulate 5-HT transmission. Direct CB1R agonists modulate DR 5-HT neural firing in brain slices (Mendiguren and Pineda, 2009) and in a bidirectional manner in vivo (Bambico et al., 2007), inhibit 5-HT reuptake ex vivo (Johnson et al., 1976; Steffens and Feuerstein, 2004) and decrease 5-HT synthesis in vivo (Moranta et al., 2004). Pharmacological or genetic deactivation of FAAH also stimulates DR 5-HT neural firing likely by increasing endocannabinoid-CB1R signaling (Bambico et al., 2010a; Gobbi et al., 2005). Interestingly, repeated CB1R activation does not seem to induce tolerance to its modulatory action on monoamine neurons, including 5-HT activity, in contrast to its many other cannabimimetic effects (Esteban and García-Sevilla, 2011). This could be related to progressive modifications in the function of monoamine-regulating presynaptic or postsynaptic receptors, such as 5-HT1A (upregulated) and α2-adrenoceptors (downregulated) (Esteban and García-Sevilla, 2011). Consistent with these 5-HT-augmenting mechanisms, synthetic cannabinoid agonists (Bambico et al., 2007) and endocannabinoid enhancement through FAAH inhibition (Gobbi et al., 2005) elicit antidepressant and anxiolytic effects detected in a wide range of behavioral tests and animal models (Bambico and Gobbi, 2008; Bambico et al., 2009a). Since few studies have been undertaken to test the antidepressant-like property of the phytocannabinoid delta-9-THC and its effect on 5-HT neurotransmission, here we examined the effects of single and repeated delta-9-THC administration on behavioral despair in the forced swim test (FST). We then examined whether this would be paralleled by neurobiological adaptations known to be associated with antidepressant activity, including the modulation of 5-HT neural firing activity in the DR and enhanced tonic hippocampal 5-HT1A receptor activity, a common neurobiological hallmark of antidepressant treatments (Haddjeri et al., 1998). 2. Materials and methods 2.1. Maintenance and preparation of animals The experiments were mainly carried out on male adult Sprague– Dawley rats (Charles Rivers, Ste. Constant, Quebec, Canada) weighing approximately 300 g. All animals were kept in pairs or trios in

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standard polycarbonate cages and maintained under standard conditions (12:12 light–dark cycle, lights on at 07:30; temperature at 20± 2 °C; 50–60% relative humidity, ad libitum access to food and water). All experiments were initiated after 1 week of acclimatization, and a 30–60 minute habituation to the testing laboratory was observed prior to each experimental run. All procedures were undertaken in compliance to the standards and ethical guidelines mandated by the Canadian Institutes of Health Research and the Canadian Council on Animal Care. 2.2. Drugs The dose of delta-9-THC (1 mg/kg) used in this study was chosen on the basis of initial intravenous administration results in electrophysiological experiments. This intraperitoneal delta-9-THC dose of 1 mg/kg corresponded to 10 times more than the minimum pharmacologically active intravenous dose (0.1 mg/kg). Moreover, a dose range of 1.0–1.5 mg/kg also represents a dose that has been reported to induce changes in intracellular transduction cascades, mRNA and protein expression, as well as mild to moderate physiological and behavioral reactions in rodents (Butovsky et al., 2005; Derkinderen et al., 2003; Nahas et al., 2002a). Furthermore, this amount is the usual dose contained in about 2 puffs (“hit”) of a marijuana joint that generally produces mild to moderate psychoactive effects (common dose of 0.67 g in humans). All drugs except for the CB1R antagonist/inverse agonist rimonabant (SR141716A, a kind gift from Dr. Daniele Piomelli) and the antidepressant citalopram HBr (kindly provided by Lundbeck, Copenhagen, Denmark) were obtained from Sigma-Aldrich Canada Ltd. Rimonabant and the CB1R/CB2R agonist delta-9-THC were dissolved in 5% Tween 80, 5% polyethylene glycol and 90% saline (0.9% NaCl solution) to a final concentration of 3 mg/ml. All other drugs were dissolved in physiological saline (0.9% NaCl solution), the 5-HT1A receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]N-(2-pyridyl)cyclohexanecarboxamide (WAY100,635) was dissolved to a final concentration of 75 μg/ml, and citalopram HBr to 30 mg/ml. Chloral hydrate was the anesthesia used in electrophysiological experiments. The pH of vehicles and solutions was adjusted to 7.2. 2.3. Effect of delta-9-THC on coping behavior in the forced swim test (FST) The FST examines the dynamics of transition from an active to a passive mode of coping in an inescapable water-filled bin. An enhancement of immobility normally ensues after exposure, a phenomenon argued to reflect learned behavioral despair, a depressivelike behavior that is prevented by antidepressant treatment (Porsolt et al., 1977). First, rats were each immersed for a 15 minute-pre-test in Plexiglas cylindrical bins (20 cm diameter, 50 cm high) filled with water (at 25–27 °C) to a depth of 20 cm, which did not allow the tail and hind paws to touch the floor of the bin. The main test was conducted 24 h later wherein rats were re-exposed for 5 min under conditions identical to the pre-test, and during which the frequency and duration of immobility, swimming and climbing episodes were encoded. We used an automated behavioral tracking system (Videotrack system, View Point Life Sciences, Montreal, Quebec, Canada) in this test as with all subsequent behavioral tests. For single drug administrations, rats received intraperitoneal injections of either delta9-THC (1 mg/kg), the vehicle or citalopram (10 mg/kg) 45 min before the main test. For repeated administrations, delta-9-THC, citalopram or the vehicle, was injected once daily for 4 days. On the 5th day, drugs were injected 5 h and 45 min prior to the main test (modified from Page et al. (1999)). In addition, some animals receiving delta9-THC (1 mg/kg) were injected with rimonabant (1 mg/kg, intraperitoneal) 15 min prior to each delta-9-THC administration. All tests were conducted toward the end of the light phase, in a dim environment and under minimal anxiogenic conditions (Kelliher et al., 2000). Custom-made plates arrayed with infrared light-emitting diodes

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were suspended above the bins. Infrared light-sensitive CCD cameras were used to capture and store images. After behavioral recording, the animals were rescued using a plastic grid and caged near a heat source. The videotrack system was initially calibrated so that a rat was considered immobile when making only those movements necessary to keep its head above water, swimming when limbs exert slow movements, and climbing when the rat engages in more forceful struggling. Ambulatory activity (total distance traveled in cm) in the open field test (OFT) was also analyzed. The OFT was carried out before the FST since results from the latter could be influenced by changes in locomotor activity. Rats were each placed at the center of a black-painted open field arena (80 × 80 × 15 cm) and left to explore the whole field for 5 min under low lighting, as previously described (Bambico et al., 2007). 2.4. Electrophysiological assessment of 5-HT transmission 2.4.1. Preparation for electrophysiological procedures In vivo single-unit extracellular recordings of DR 5-HT neurons were performed as previously described (Bambico et al., 2007; Gobbi et al., 2005). First, rats were anesthetized with chloral hydrate (chloral hydrate, 400 mg/kg, intraperitoneal) then mounted in a stereotaxic frame (David Kopf Instruments, Tujunga, California) with the skull positioned horizontally (incisor bar at − 3.3 mm). Anesthesia was confirmed by the absence of nociceptive reflex reaction to a tail or a paw pinch and of an eye blink response to pressure. To maintain a full anesthetic state characterized by the absence of a nociceptive reaction to a paw/tail pinch and eye blink response to pressure, supplemental doses of chloral hydrate (100 mg/kg, intraperitoneal) were periodically administered. Body temperature was maintained at 37 ± 0.5 °C throughout the experiment using a thermistorcontrolled heating pad (Seabrook Medical Instrument, Inc.). Recordings were carried out using single-barrelled (R&D Scientific Glass, Spencerville, MD) or microiontophoresis multi-barrelled (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 2 M NaCl and their tips were broken down to diameters of 1 to 3 μm for singlebarrelled and 10–15 μm for multi-barrelled ones. The impedances ranged from 1.5 to 2.5 and 2 to 6 MΩ, respectively. Using a hydraulic micropositioner (model 650; David Kopf Instruments, Tujunga, California), the electrode was advanced slowly into the brain structure, guided by coordinates from Paxinos and Watson (1986), until a neuronal signal was isolated. In order to minimize the probability of missing slow-spiking neurons, the speed of descent was clamped to approximately 0.15 mm/min. For multiple single-unit recordings, three to five electrode descents were achieved to maximize sampling without introducing considerable tissue damage. Single-unit activity was recorded as large-amplitude action potentials captured by a software window discriminator, amplified by a Tennelec (Oakridge, TN) TB3 MDA3 amplifier, post-amplified and band-pass 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 by Spike2 software version 5.20 for Windows PC (Microsoft, Seattle, WA). An Npi electronic Gmbh microiontophoresis 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 intravenous drug administration, a catheter was inserted into the lateral tail vein prior to electrophysiological recording and the maximum volume administered for cumulative intravenous doses was 0.4 ml. Drug response was considered inhibitory or excitatory if changes in neural activity exceeded 10% of the basal firing rate as

described in Bambico et al. (2007). At the end of each recording session, the recording site was marked by iontophoretic ejection (1–10 mA, negative current for 10 min) of Pontamine Sky Blue for later histological verification of recording sites when needed. All recordings were carried out between 14:00 and 22:00. 2.4.2. Extracellular recordings of spontaneous DR 5-HT neural firing activity The DR is the main source of 5-HT innervation in the brain. To record from single-unit 5-HT neurons in the DR, an incision was made on the scalp and the periosteum moved aside, then, a burr hole was drilled on the cranial midline subtending regions above the entire rostrocaudal medial extent of the DR presumed richest in 5-HT neurons (Descarries et al., 1982). The electrode was lowered into the region of the DR (1.2 mm anterior to interaural zero on the midline, 5.0 to 6.5 mm from the dura mater, which is just beneath the Sylvian aqueduct). Under physiological conditions, spontaneously active 5-HT neurons exhibit characteristic electrophysiological properties distinguishable from non-5-HT neurons. These 5-HT neurons exhibit a slow (0.1–4 Hz) and a prominently regular firing rate (coefficient of variation, C.O.V., ranges from 0.12 to 0.87), a broad biphasic (positive–negative) or triphasic waveforms (0.8–3.5 ms; 1.4 ms first positive and negative deflections) (Allers and Sharp, 2003; Bambico et al., 2007; Van der Maelen and Aghajanian, 1983). Although, these criteria may vary in response to pharmacological or environmental manipulations (Bambico et al., 2009b), some spike features, i.e., waveform shape and spike duration, have been shown to be stable across conditions, and are therefore reliable indicators for 5-HT neurons (Urbain et al., 2006; Van der Maelen and Aghajanian, 1983). When the regularity of firing was apparently altered by drug exposure, i.e., exceeding C.O.V. 0.87, neurons were rigidly discriminated based on firing rates, spike shape and duration. 2.5. Extracellular recordings and microiontophoresis from the CA3 hippocampus The animals were subjected to an injection schedule similar to the one conducted for the FST. The microiontophoresis procedure was modified after Gobbi et al. (2001) and Haddjeri et al. (1998). The multi-barrelled micropipette was lowered into the CA3 regions of the dorsal hippocampus (2 to 3 mm lateral and 2.5 to 2.7 mm posterior to bregma, Paxinos and Watson, 1986). The side-barrels had impedances ranging from 50 to 150 MΩ and contained quisqualic acid (1.5 mM in 400 mM NaCl, pH 8) and 2 M NaCl solution for automatic current balancing. Since most hippocampal pyramidal neurons are not spontaneously active under chloral hydrate anesthesia, prolonged low-current quisqualate ejections (from −2 to −5 nA) were introduced in order to activate them within their physiological firing rates (8–15 Hz) and were retained with a current of +10 nA. Tonic 5-HT1A receptor activity was assessed by intravenous injections of cumulative doses of WAY100635 (up to 100 μg/kg). 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 (Kandel and Spencer, 1961). Pyramidal neural response to systemic drug application was expressed as percentage increase/ decrease from pre-drug (baseline) activity. At the end of electrophysiological experiments, animals were euthanized and the brains were harnessed and stored in paraformaldehyde. When needed, coronal brain slices through regions of interests were prepared to verify the location and extent of electrode lesions under a light microscope. 2.6. Data analyses and statistics Data are presented as mean ± standard error of the mean (SEM). All data were organized and analyzed using SPSS version 17 (SPSS Inc., Chicago, Illinois), Sigma Plot version 11.0 (Systat Software Inc.,

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inert response. Fig. 1A presents a scatterplot of the computed percentage change (increase or decrease) in the spontaneous firing rate of these neurons. The excitatory responses were spread across low and high doses, and were maximal at 1 mg/kg. The inhibitory responses were mainly produced by doses smaller than 0.6 mg/kg and the maximal response was at 0.2 mg/kg. Inert responses were equally distributed between low and high dose ranges. Chi-square analysis failed to reveal any significant difference in the relative percentage of neurons exhibiting these different response profiles (χ 22 = 3.5, p = 0.17). It was also less likely that carry-over effects from cumulative dosing significantly influenced the distribution of responses since 35 neurons that received single intravenous doses also yielded similar response profiles, with 37.14% (n = 13) being excited, 25.71% (n = 9) inhibited and 37.14% (n = 13) unaffected (data not shown). Likewise, there was no significant difference in the relative percentage of neurons exhibiting these different response profiles (χ 22 = 0.91, p = 0.634). Recorded 5-HT neurons, regardless of their response, exhibited identical electrophysiological characteristics and were all recorded from the rostrocaudal midline extent of the DR nucleus. In an attempt to determine which receptor subsystems were responsible for the excitatory and inhibitory responses, rimonabant (1 mg/kg) or capsazepine (0.1 mg/kg) was intravenously administered following a delta-9-THC-induced increase or decrease in 5-HT neural firing activity. In all neurons tested (n = 3), the excitatory response was attenuated by rimonabant but not by capsazepine, suggesting that delta-9-THC-induced excitations were instigated by CB1R activation. On the other hand, the inhibitory response was only partially reversed by rimonabant in 1 out of 3 neurons, and was not at all sensitive to capsazepine, indicating a non-cannabinoid CB1R and a non-vanilloid mechanism. Interestingly, the inhibitory

San Jose, California) and Excel 2007 (Microsoft Office). After testing for assumptions of normality of data distribution and of homogeneity of variance, behavioral data analyses were accordingly conducted with general linear model, mixed design ANOVA or with one-way ANOVA. Electrophysiological data were submitted to Mann–Whitney U-tests for between-group comparisons, to two-way mixed design ANOVA (drug × current), when accounting for repeated microiontophoretic current ejections, or to chi-square (X 2) test. Tukey's honestly significant difference (HSD) test was used for multiple post hoc comparisons. Probability value of p ≤ 0.05 was considered to be statistically significant. 3. Results 3.1. Intravenous administration of delta-9-THC produces a complex response from DR 5-HT neurons We have previously shown that the antidepressant-like activity of the CB1R agonist WIN55,212-2 (Bambico et al., 2007) and the FAAH inhibitor URB597 (Gobbi et al., 2005) may be driven by a modulatory action on DR 5-HT neurons. We therefore examined whether delta-9THC evokes an identical response from these neurons. We first assessed the effect of intravenous administrations of different doses of delta-9-THC (0.1 to 1.5 mg/kg), which yielded a complex response profile. Eighty-six individual dose–responses were recorded from a pool of DR 5-HT neurons that each received a maximum of 3 cumulative doses. Zero dose corresponded to saline injection. We were able to identify three response profiles to delta-9-THC within this dose range: 25.58% (n = 22) showed excitation (≥10% of baseline), 32.56% (n = 28) inhibition (≥10% of baseline) and 41.86% (n = 36)

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Fig. 1. Acute intravenous administration of variable doses of delta-9-THC elicited a complex response profile from dorsal raphe (DR) 5-HT neurons. (A) Top illustration portrays a coronal brain section (Paxinos and Watson, 1986) containing the dorsal raphe (DR) nucleus, just beneath the Sylvian aqueduct (Aq, gray shade). The boxed area encompassing the DR represents the location where most putative 5-HT neurons were found. The waveform represented is a typical action potential (broad, biphasic, positive–negative spike) of a putative 5-HT neuron as seen on the oscilloscope. The bottom graph depicts a scatterplot of the evoked changes (% of baseline, ordinate) in the neural firing activity of putative 5-HT neurons that each received a maximum of 3 cumulative doses (all delta-9-THC doses are plotted along the abscissa). Excitatory, inhibitory and inert responses were distributed within a narrow dose range from 0.1 mg/kg to 1.5 mg/kg. N=22-36/group. (B) The top and bottom panels each present a response of a 5-HT neuron to different intravenous drug challenges as indicated by arrows and corresponding labels (number above arrows indicates the intravenous dose administered in mg/kg). In each, the first and third channels plot spike and event traces, respectively. Each waveform or line counts as an individual action potential. The second channel plots representative firing rate histograms. Each bar represents the number of spikes (ordinate) per 10 s (abscissa, time in seconds). Top panel: While injection with the vehicle did not elicit a significant change in 5-HT neural firing rate, delta-9-THC (1 mg/kg as indicated) increased it. An elevated firing was maintained after an injection with the vanilloid (TRPV1) antagonist capsazepine (0.1 mg/kg as indicated). Subsequent administration of the CB1R antagonist/inverse agonist rimonabant (1 mg/kg as indicated) reversed the delta-9-THC-evoked increase in neural firing rate. Bottom panel: Delta-9-THC (1 mg/kg as indicated) attenuated 5-HT neural firing activity. Subsequent injection with either rimonabant (1 mg/kg as indicated) or capsazepine (0.1 mg/kg as indicated) was without any effect. N=3/group.

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effect of anandamide on a number of DR 5-HT neurons ex vivo was also demonstrated elsewhere to have been mediated by a noncannabinoid CB1R mechanism (Mendiguren and Pineda, 2009). Corresponding representative integrated firing rate histograms are shown in Fig. 1B. 3.2. Intraperitoneal administration of delta-9-THC modulates spontaneous DR 5-HT neural activity We then administered 1 mg/kg of delta-9-THC intraperitoneally, which corresponded to 10 times more than the minimum pharmacologically active intravenous dose (0.1 mg/kg). A single intraperitoneal administration of this dose of delta-9-THC had a minimal effect, and failed to significantly enhance the mean spontaneous 5-HT neural firing rate after 1.5 h of continuous neuro-electrophysiological sampling. Increasing the dose to 2 and 4 mg/kg similarly yielded nonsignificant elevations in mean 5-HT neural firing rate (Fig. 2). Higher doses were not tested in order to avoid non-selective effects of delta9-THC. We next tested the effect of repeated administration (5 days) of 1 mg/kg of delta-9-THC (intraperitoneal) on DR 5-HT neural firing activity, which yielded a 60% increase in mean spontaneous 5-HT neural firing rate after 1.5 h of electrophysiological recordings (p b 0.05). This increase was blocked by the co-application of rimonabant (1 mg/kg, intraperitoneal) and was therefore mediated by enhanced CB1R activity (Fig. 2). This increased mean firing after a repeated regimen may be due to a change in the proportion of excited, inhibited and non-responding neurons, and may represent longterm neuroplastic or synaptic adaptive modifications maintained by prolonged CB1R activation. 3.3. Delta-9-THC elicits an antidepressant-like coping response in the forced swim test (FST) We assessed the effect of delta-9-THC on active and passive stresscoping behaviors in the FST, a well-established and reliable first-stage behavioral screen for antidepressant activity, using the intraperitoneal dose regimen used for the electrophysiological experiment. Mixeddesign ANOVA was performed to determine if there was a difference among the effects of treatment (drug and vehicle injections) in the

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Delta-9-THC (mg/kg, intraperitoneal) Fig. 2. Repeated delta-9-THC intraperitoneal administration enhanced dorsal raphe (DR) 5HT neural firing activity. Left panel: Mean 5-HT neural firing rates (bars in Hz±SEM) following single intraperitoneal administration of delta-9-THC at 1, 2 or 4 mg/kg were not significantly different from those produced by single intraperitoneal administration of the vehicle. Right panel: Repeated intraperitoneal administration of delta-9-THC (1 mg/kg, 5 days) significantly increased mean spontaneous 5-HT neural firing rate (bars in Hz± SEM), in comparison to repeated (5 days) vehicle treatment. *pb 0.05. Co-administration of the CB1R antagonist/inverse agonist rimonabant (1 mg/kg, intraperitoneal) with repeated intraperitoneal delta-9-THC (1 mg/kg, 5 days) prevented the delta-9-THC-induced increase in mean spontaneous 5-HT neural firing (bars in Hz±SEM). N=21-53 neurons/group. Top illustration represents a coronal brain section (Paxinos and Watson, 1986) containing the DR nucleus (boxed), and a typical 5-HT waveform.

amount of change in the frequency (number of episodes) and duration (total time in seconds) of coping behaviors (immobility, swimming and climbing) in the FST. Under the single administration schedule, delta-9-THC (1 mg/kg, intraperitoneal) was not sufficient to elicit any difference in either the frequency (Fig. 3, top left panel) or total duration (Fig. 3, top right panel) of coping behaviors in comparison to control (vehicle). After a repeated administration schedule (5 days), analysis of frequency revealed significant main effects of treatment (F3,27 = 3.44, p b 0.05), behavior (F2,54 = 117.94, p b 0.01), as well as a significant interaction between these two factors (F6,54 = 2.60, p b 0.05) (Fig. 3, lower left panel). Pairwise multiple comparisons (Tukey's test) indicated that group differences were only evident for swimming behavior, where both delta-9-THC and the antidepressant citalopram yielded greater frequencies compared to control (p b 0.05) (Fig. 3, lower left panel). Note that swimming and climbing episodes apparently increased after repeated administrations compared to single administrations, which was likely an effect of repeated handling and/or injections. Following repeated (5 days) administrations, a significant main effect of behavior (F2,54 = 37.21, p b 0.01) but not of treatment, along with a significant treatment-behavior effect (F6,54 = 5.12, p b 0.01), on total duration was calculated (Fig. 3, lower right panel). Pairwise multiple comparisons (Tukey's test) indicated that group differences occurred within all behaviors. Citalopram yielded a significant decrease in mean immobility duration when compared to control (p b 0.01). This antidepressant-induced reduction in immobility was also instigated by delta-9-THC (p b 0.01), an effect that was nullified when co-administered with rimonabant (p = 0.36). Only citalopram treatment led to a significant enhancement in swimming behavior (p b 0.05), while delta-9-THC elicited instead an increase in climbing behavior (p = 0.05). This effect of delta-9-THC was abrogated by cotreatment with rimonabant (p = 0.99), and was therefore likely mediated by enhanced CB1R activity (Fig. 3, lower right panel). Acute or repeated delta-9-THC (1 mg/kg, intraperitoneal) treatment was without influence on locomotor activity (distance traveled, data not shown). 3.4. Delta-9-THC enhances hippocampal serotonergic 5-HT1A transmission We employed in vivo extracellular recordings to monitor changes in the activity of pyramidal neurons in the CA3 region of the dorsal hippocampus (Fig. 4, boxed area of left diagram) in response to cumulative systemic administration of the 5-HT1A receptor antagonist WAY100635 (25–100 μg/kg, intravenous). Under control (normal physiological) conditions, WAY100635 is normally without any significant effect on pyramidal activity within the dose range used here. Under conditions of increased tonic 5-HT1A receptor, such as resulting from prolonged antidepressant treatment, WAY100635 has been shown to acquire the capacity to increase (disinhibit) hippocampal pyramidal firing activity (Besson et al., 2000; Haddjeri et al., 1998). Our data indicated that single intraperitoneal administration of either the antidepressant citalopram (10 mg/kg) or delta-9-THC (1 mg/kg), compared to control (vehicle), failed to bestow WAY100635 the capacity to disinhibit hippocampal pyramidal neurons, suggesting an unmodified tonus on 5-HT1A receptors (data not shown). However, repeated (5 days) citalopram treatment increased the excitatory response of hippocampal CA3 pyramidal neurons to WAY100635, an effect that was positively related to dose (Fig. 4). Repeated (5 days) intraperitoneal administration of delta-9-THC (1 mg/ kg) also yielded a similar effect (Two-way ANOVA; treatment group: F2,24 = 11.96, p b 0.01; current: F4,24 = 2.21, p = 0.09; group × current: F4,24 = 2.34, p = 0.05; AUC, repeated: 106.10% vs. vehicle, p b 0.05; Fig. 4). There was a significant between-groups difference between repeated vehicle treatment and repeated citalopram or repeated delta-9-THC (p b 0.01 and p b 0.05, respectively). Across currents,

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Fig. 3. Effect of delta-9-THC on coping behaviors in the forced swim test (FST). Single intraperitoneal administration of delta-9-THC (1 mg/kg), in comparison to vehicle, failed to significantly alter the frequency (# of episodes ± SEM, upper left panel) or duration (seconds ± SEM, upper right panel) of immobility (white bars), swimming (gray bars) and climbing (black bars) behaviors. Single intraperitoneal administration of the antidepressant citalopram (10 mg/kg) also did not change the frequency and duration of immobility, swimming and climbing behaviors in the FST. In comparison to repeated (5 days) vehicle administration, repeated intraperitoneal administration of delta-9-THC (1 mg/kg, 5 days) significantly increased the frequency (# of episodes ± SEM, lower left panel) of swimming (gray bars) behavior, without significantly altering that of immobility (white bars) or climbing (black bars). This effect was similarly elicited by the repeated intraperitoneal administration of citalopram (10 mg/kg, 5 days) (lower left panel). In comparison to repeated (5 days) vehicle administration, repeated intraperitoneal administration of delta-9-THC (1 mg/kg, 5 days) significantly decreased the mean duration (seconds ± SEM) of immobility (white bar), and increased that of climbing (black bar), without modifying that of swimming (gray bar) (lower right panel). Repeated citalopram treatment (10 mg/kg, 5 days) also yielded a significant decrease in the mean duration (seconds ± SEM) of climbing (black bar), an increase in swimming (gray bar), and without a marked effect on climbing (black bar) (lower right panel). N=7-9/group. *p b 0.05.

these significant differences between vehicle and citalopram or delta9-THC were pronounced at 75 nA and 100 nA. These effects of repeated citalopram and delta-9-THC treatments point to an enhancement in the tonic activity of hippocampal 5-HT1A receptor. 4. Discussion

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In this study, we present experimental evidence that delta-9-THC could elicit antidepressant-like activity, and this behavioral effect is paralleled by an increase in spontaneous dorsal raphe (DR) nucleus 5-HT neural activity and an enhanced tonic activity of 5-HT1A

receptors in the dorsal hippocampus, which are neurobiological signatures of antidepressant action. Our initial electrophysiological experiments revealed that intravenous challenges of different doses of delta-9-THC produce a complex response. Excitatory, inhibitory and inert responses were exhibited within a narrow dose range among different subsets of 5-HT neurons. Both excitatory and inhibitory responses appeared to assume bellshaped distributions. The maximally effective intravenous excitatory dose (1 mg/kg), which was tenfold the minimum active dose tested, was used for single intraperitoneal administrations. This intraperitoneal dose failed to significantly modify the mean discharge rate of

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WAY100635 (microgram/kg, intravenous) Fig. 4. Repeated intraperitoneal treatment with delta-9-THC enhanced the tonic activity of hippocampal (postsynaptic) 5-HT1A heteroreceptors. Left panel: A diagram of a coronal section through the CA3 region of the dorsal hippocampus (boxed) whence pyramidal neurons were obtained. Shown above is a typical trace of a complex spike train exhibited by pyramidal neurons. Right panel: The line graph describes the excitatory (disinhibitory) responses of pyramidal neurons (data points in % baseline ± SEM, n=15/group) to cumulative intravenous doses of the 5-HT1A antagonist WAY100635 (0–100 μg/kg). In comparison to repeated (5 days) intraperitoneal administration of the vehicle (white circles), repeated (5 days) intraperitoneal treatment of either the antidepressant citalopram (10 mg/kg, gray squares) or delta-9-THC (1 mg/kg, black triangles) led to an enhanced excitatory response of pyramidal neurons that was positively related to the dose of WAY100635. **p b 0.01 between-groups difference, citalopram vs. vehicle; *p b 0.05 between-groups difference, delta-9-THC vs. vehicle.

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DR 5-HT neurons. Higher intraperitoneal doses of up to 4 mg/kg also did not yield significant effects. This weak response profile associated with acute/single delta-9-THC treatment may be ascribed to its partial agonist activity at CB1R. Further research on the role of delta-9THC's pharmacological properties in monoaminergic transmission and emotional processes is warranted. Recent evidence suggests that delta-9-THC could act both as a CB1R partial and full agonist, depending on whether CB1R is localized in GABAergic or glutamatergic synapses (Laaris et al., 2010). Interestingly, repeated delta-9-THC treatment (1 mg/kg, 5 days) yielded a significant elevation in the mean discharge rate of DR 5HT neurons, an effect that appeared to be CB1R-dependent since it was reversed by co-administration with the CB1R antagonist/inverse agonist rimonabant. This modulation in 5-HT neural activity has been previously observed with the CB1R agonist WIN55,212-2 or the endocannabinoid enhancing FAAH inhibitor URB597, demonstrated in vivo (Bambico et al., 2007; Gobbi et al., 2005; Palazzo et al., 2006) and with the CB1R agonist arachidonoyl-2-chloroethylamide (ACEA) ex vivo (Mendiguren and Pineda, 2009). Conversely, CB1R antagonism by rimonabant and AM251, depresses 5-HT neural activity in brain slices (Mendiguren and Pineda, 2009). Delta-9-THC has also been shown by others to increase in vivo the activity of NE neurons of the locus coeruleus nucleus (Muntoni et al., 2006) and dopaminergic neurons of the ventral tegmental area (French et al., 1997), which could feedback onto 5-HT activity via their reciprocal connectivity with the DR (Guiard et al., 2008). In addition, glucocorticoids and corticotropin-releasing factor could influence 5-HT activity (Lowry et al., 2000). It is therefore plausible that delta-9-THC's capacity to activate the hypothalamic–pituitary–adrenocortical (HPA) axis (Steiner and Wotjak, 2008) may contribute to these effects on monoaminergic activity. The behavioral significance of these effects on 5-HT and on other monoaminergic neurons, as well as on HPA activation, and their differential contributions to stress adaptation, could be an important theme of future investigations. Interestingly, repeated administration of delta-9-THC (1 mg/kg) reduced immobility in the forced swim test (FST), resembling the action of the antidepressant citalopram and indicated effective recovery from a passive mode of coping or behavioral despair, a key feature of depression. This reactivity was determined to be CB1R-dependent because co-administration with the CB1R antagonist/inverse agonist rimonabant nullified the effect. Moreover, acute administration of this dose produced classical signs of CB1R activation, including analgesia and catalepsy (data not shown) that were unlikely to have influenced immobility in the FST, and may have affected a different neural circuit altogether. Both acute and repeated administrations of this dose were without marked influence on locomotor activity. Others have reported similar findings with delta-9-THC in the FST (El-Alfy et al., 2010; Moreira et al., 2008), as well as, using other behavioral models such as olfactory bulbectomy (OBx) (ElBatsh et al., 2009; Rodríguez-Gaztelumendi et al., 2009) and tail suspension test (TST) in mice (El-Alfy et al., 2010). These further corroborate the antidepressant-like properties reported of other CB1R agonists (Bambico et al., 2007; Hill and Gorzalka, 2005) and endocannabinoid enhancers, such as FAAH inhibitors and endocannabinoid reuptake inhibitors (Adamczyk et al., 2008; Bortolato et al., 2007; Gobbi et al., 2005). Conversely, genetic CB1R (CNR1) deletion (Aso et al., 2008; Martin et al., 2002; for review, Valverde and Torrens, 2012) or CB1R antagonism (Beyer et al., 2010) has been found to lead to depressive-like behaviors in animal models. Clinical findings on rimonabant (Acomplia) have likewise ascertained these risks for depression, anxiety and suicidality (Mitchell and Morris, 2007). In contrast to our observations, Egashira et al. (2008) reported that delta-9-THC administration resulted in an increase rather than a decrease in immobility in the FST. This discrepancy may be explained by several factors. This other study employed ddY mice; such a difference in species/strain could influence the regional

distribution, density or responsiveness of cerebral CB1Rs (Haller et al., 2007; Hoffman et al., 2005; Hungund and Basavarajappa, 2000; McPartland et al., 2007). Also, the FST protocol employed involved movement detection by way of a magnet attached to one of the animal's forelimbs. Notably, the doses used (2 and 6 mg/kg) were higher than that administered here, suggesting that at higher doses delta-9THC could acquire depressogenic properties. On the other hand, the observation that antidepressant-like reductions in immobility were only detected after repeated but not after a single treatment was similar to that previously observed with the FAAH inhibitor URB597 (Gobbi et al., 2005). Worth noting is that delta-9-THC specifically enhanced climbing and not swim behavior, reflective of the behavioral profile of NE-acting agents such as tricyclic antidepressants (TCAs), in the FST. Interestingly, an identical profile was reported to proceed from a protracted administration of the CB1R agonist HU-210 (Morrish et al., 2009). On the other hand, we have previously shown that short-term (sequential 3× over 24 h) treatment with the CB1R agonist WIN55,212-2 enhances swim rather than climbing behavior, mimicking the effect of the SSRI citalopram, a 5-HT-acting antidepressant (Bambico et al., 2007). We cannot therefore exclude that cannabinoid agonists act on both swim and climbing behaviors, depending on the treatment schedule employed. Indeed, we also noted that repeated WIN55,212-2 administration achieves tolerance of swim behavior and sensitization of climbing (Bambico et al., 2008). We also showed that repeated but not single delta-9-THC administration resulted in an increase in the tonic activity of hippocampal 5-HT1A receptors, a postsynaptic adaptation associated with antidepressant action. The lack of effect following single delta-9-THC administration may have likely accounted for the difficulty in detecting acute antidepressant-like activity in the FST. The positive effect of repeated delta-9-THC treatment was recapitulated by repeated citalopram administration, as well as by repeated URB597 administration, as previously demonstrated (Bambico et al., 2010a). This response is known to be elicited by both 5-HT and NE-acting antidepressants, and are therefore suggested to be a mechanism common among all classes of antidepressants (Besson et al., 2000; Haddjeri et al., 1998). Earlier, we have demonstrated that FAAH-deficient mice, which exhibit enhanced anandamide-CB1R signaling, also show antidepressant-like behavior in the FST paralleled by enhanced tonic hippocampal 5-HT1A activity (Bambico et al., 2010a). We hypothesized that this neurophysiological adaptation could have contributed in conveying the depressive/anxiety-resistant phenotype of these mice. The behavioral and neurophysiological potency of repeated delta9-THC administration may be explained by neuroplastic changes incurred over time. Cannabinoids have been implicated in different forms of short-term and long-term plasticity that include depolarizationinduced suppression of excitation or inhibition and long-term depression (for review, Heifets and Castillo, 2009; Zhu, 2006). This may involve, at least in part, 5-HT1A-associated synapses. Indeed, CB1R agonists could increase hippocampal 5-HT1A binding and mRNA expression (Zavitsanou et al., 2010), and 5-HT1A is found to be involved in the anxiolytic-like action of delta-9-THC and AM404 (Braida et al., 2007). On the other hand, we cannot rule out that the effects observed here may also be ascribed in part to delta-9-THC's agonistic action on CB2Rs. This is in light of recent evidence for CB2R's central expression (Gong et al., 2006). It has recently been demonstrated that overexpression of CB2R conveys resistance to depression (García-Gutiérrez et al., 2010) and intracerebroventricular microinjection of CB2 antisense oligonucleotide into the mouse brain led to an anxiolytic effect (Onaivi et al., 2008). More research is needed to understand the relative contribution of CB1R and CB2R to delta-9-THC's emotional effects. Overall, our electrophysiological data are in agreement with reported increases in the turnover or postsynaptic release of 5-HT or NE by delta-9-THC and CB1R agonists detected in the prefrontal cortex (Jentsch et al., 1997; Page et al., 2008), as well as by endocannabinoid enhancers detected in the hippocampus (Bambico and

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Gobbi, 2008; Gobbi et al., 2005), which is recapitulated in FAAHdeficient mice (Cassano et al., 2011). Furthermore, CB1R agonists also are able to block the reuptake of 5-HT and dopamine (DA), increasing their synaptic content (Steffens and Feuerstein, 2004). However, conflicting data have also been presented with delta-9-THC having been reported to increase 5-HT content while inhibiting its release in the ventral hippocampus (Egashira et al., 2002) and nucleus accumbens (Sano et al., 2008). The CB1R antagonist/inverse agonist rimonabant, at doses greater than 1 mg/kg, has also been reported to acutely enhance the efflux of 5-HT, NE and DA efflux in the medial prefrontal cortex, and those of NE and DA in the nucleus accumbens (Tzavara et al., 2003). In summary, the data at hand indicate that delta-9-THC after repeated administration and at a relatively low dose (1 mg/kg) could convey antidepressant activity via enhancement of 5-HT transmission in the DR and hippocampus, a characteristic feature comparable to other classes of antidepressants. These results stand out against previous findings on the potential depressogenic and anxiogenic effects of cannabinoids resulting from their long-term consumption especially during critical developmental periods such as adolescence (Bambico and Gobbi, 2008; Bambico et al., 2010b). Altogether, these findings suggest that delta-9-THC and other CB1R agonists may have dual-opposite effects on mood modulation depending upon the extent or duration of use, dose, and age of the user. Indeed, the acute dual-opposite or mixed effect on 5-HT transmission we reported is suggestive of very complex psychoactive effects associated with delta-9-THC. This then could represent a potential basis for the experienced variable effects among the population, ranging from aversive and psychotomimetic consequences to mood elevation. Further studies need to be carried out to completely understand how this variable/dual effect profile on 5-HT transmission is linked to subjective experience and emotional behavior. Statement of disclosure and conflicts of interest The authors do not have any conflicts of interest or any financial involvements or other activities to disclose, which may potentially bias the conduct, interpretation or presentation of this work. Acknowledgment We would like to extend our gratitude to Mr. Herculano Santos and Mr. Normand Champagne for their technical assistance in the handling and maintenance of the animals during the course of the experiments. This work was supported by grants from the Fonds de la Recherche en Santé du Québec (FRSQ) (G.G.) and fellowships from the McGill University Health Center, McGill University Faculty of Medicine and FRSQ (F.R.B.). References Adamczyk P, Golda A, McCreary AC, Filip M, Przegaliński E. Activation of endocannabinoid transmission induces antidepressant-like effects in rats. J Physiol Pharmacol 2008;59(2):217–28. Allers KA, Sharp T. Neurochemical and anatomical identification of fast- and slow-firing neurones in the rat dorsal raphe nucleus using juxtacellular labelling methods in vivo. Neuroscience 2003;122:193–204. Aso E, Ozaita A, Valdizán EM, Ledent C, Pazos A, Maldonado R, et al. BDNF impairment in the hippocampus is related to enhanced despair behavior in CB1 knockout mice. J Neurochem 2008;105(2):565–72. Bambico FR, Gobbi G. The cannabinoid CB1 receptor and the endocannabinoid anandamide: possible antidepressant targets. Expert Opin Ther Targets 2008;12(11): 1347–66. Bambico FR, Katz N, Debonnel G, Gobbi G. Cannabinoids elicit antidepressant-like behavior and activate serotonergic neurons through the medial prefrontal cortex. J Neurosci 2007;27:11700–11. Bambico FR, Hattan PR, Katz N, Gobbi G. Differences between fatty acid amide (FAAH inhibitors) and CB1 agonists on the modulation of antidepressant-like behaviour and serotonergic neurotransmission. 38th Annual Meeting. Washington, D.C., USA: Society for Neuroscience; 2008.. November 15–19.

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