Cannabinoids elicit antidepressant-like behavior and activate serotonergic neurons through the medial prefrontal cortex

Abstract

Preclinical and clinical studies show that cannabis modulates mood and possesses antidepressant-like properties, mediated by the agonistic activity of cannabinoids on central CB1 receptors (CB1Rs). The action of CB1R agonists on the serotonin (5-HT) system, the major transmitter system involved in mood control and implicated in the mechanism of action of antidepressants, remains however poorly understood. In this study, we demonstrated that, at low doses, the CB1R agonist WIN55,212-2 [R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl) methanone mesylate] exerts potent antidepressant-like properties in the rat forced-swim test (FST). This effect is CB1R dependent because it was blocked by the CB1R antagonist rimonabant and is 5-HT mediated because it was abolished by pretreatment with the 5-HT-depleting agent parachlorophenylalanine. Then, using in vivo electrophysiology, we showed that low doses of WIN55,212-2 dose dependently enhanced dorsal raphe nucleus 5-HT neuronal activity through a CB1R-dependent mechanism. Conversely, high doses of WIN55,212-2 were ineffective in the FST and decreased 5-HT neuronal activity through a CB1R-independent mechanism. The CB1R agonist-induced enhancement of 5-HT neuronal activity was abolished by total or medial prefrontocortical, but not by lateral prefrontocortical, transection. Furthermore, 5-HT neuronal activity was enhanced by the local microinjection of WIN55,212-2 into the ventromedial prefrontal cortex (mPFCv) but not by the local microinjection of WIN55,212-2 into the lateral prefrontal cortex. Similarly, the microinjection of WIN55,212-2 into the mPFCv produced a CB1R-dependent antidepressant-like effect in the FST. These results demonstrate that CB1R agonists possess antidepressant-like properties and modulate 5-HT neuronal activity via the mPFCv.

Figure 1.

Antidepressant-like activity of WIN in the rat FST. A, Behavioral effects of intraperitoneally administered vehicle, CIT (5 mg/kg, i.p.), DMI (10 mg/kg, i.p.), and WIN (0.05, 0.1, 0.2, 1.0, and 2.0 mg/kg, i.p.). Single injection of RIM (1 mg/kg, i.p.) 10 min before administration of a low dose of WIN (0.2 mg/kg, i.p.) blocked the antidepressant-like effect. Single injection of RIM (1 mg/kg, i.p.) 10 min before administration of a high dose of WIN (2 mg/kg, i.p.) did not modify the inert effect of WIN. Note that RIM by itself did not have any significant effect. B, The antidepressant-like effect of WIN (0.2 mg/kg, i.p.) was abrogated by pretreatment with pCPA (150 mg/kg, i.p.) 72 and 48 h before pretest. Pretreatment of pCPA by itself did not have any significant effect. All treatments were administered 23, 5, and 0.75 h before test swim according to the method of Page et al. (1999). n = 8–15 per treatment group. Bars represent mean ± SEM total time of behaviors indicated. *p < 0.05 or **p < 0.01 versus vehicle.

Figure 2.

Effect of intraperitoneal administration of WIN on DR 5-HT neurons. A, Effect of WIN on 5-HT neuronal firing activity. WIN (0.05–2.0 mg/kg, i.p.) was administered 23, 5, and 0.75 h before electrophysiological recordings. Coapplication of RIM (1 mg/kg, i.p.) 10 min before WIN (0.2 mg/kg, i.p.) blocked the increase in spontaneous 5-HT single-unit firing activity. B, Effect of WIN on the number of spontaneously active 5-HT neurons. The number of spontaneously active neurons was calculated as the number of recorded 5-HT neurons per electrode descent in each treatment group. Values at the base of each column in A denote the number of 5-HT neurons recorded. Bars represent mean ± SEM values. **p < 0.01 versus vehicle.

Figure 3.

Effect of intravenous administration of cumulative doses of WIN on DR 5-HT neurons. A–D, Integrated firing rate histograms of 5-HT neurons illustrating that low doses of WIN (0.1–0.2 mg/kg, i.v.) rapidly increased single-unit firing activity. A, This effect was reversed by RIM (1.0 mg/kg, i.v.; n = 4) but not by CPZ (0.02 mg/kg, i.v.; n = 4). B–D, High dose of WIN (0.30–0.50 mg/kg, i.v.) rapidly decreased single-unit firing activity. This effect was reversed by CPZ (0.02 mg/kg, i.v.) in two of three neurons (D) and partially reversed (B) or unreversed (C) by RIM (1 mg/kg, i.v.) in one and three neurons, respectively. 5-HT neuronal firing rate in each histogram is plotted as spikes per 10 s. Calibration bar on right side of each histogram, 1 min. The vertical lines depicted below each histogram represent the frequency of neuronal burst activity such that each tick corresponds to a burst discharge event. E, WIN (0.05–0.5 mg/kg, i.v.) produced a biphasic response profile in 5-HT single-unit activity. F, Line graphs showing that cumulative doses of WIN modulated 5-HT neuronal burst activity measured as percentage of spikes within bursts (top) and mean burst length (bottom). *p < 0.05 or **p < 0.01 vs baseline (vehicle).

Figure 4.

Effect of PFC transections on the modulation of DR 5-HT neuronal activity by intravenous administration of WIN. A, Line graph showing the modulatory effect of cumulative doses of WIN (0–0.5 mg/kg) on 5-HT single-unit firing activity after tPFC (shaded inverted triangles), ablation of mPFC (shaded squares), or latPFC (shaded upright triangles) subregions compared with sham-exposed controls (open circles). tPFC and mPFC transections abrogated the excitatory response to low doses of WIN (0.05–0.2 mg/kg, i.v.), whereas latPFC transection did not significantly reduce the excitatory response to low doses of WIN (0.05–0.2 mg/kg, i.v.). n = 3–7 animals per group. Values are expressed as mean ± SEM of increase in 5-HT unit firing rate (percentage of baseline). **p < 0.01 mPFC transection versus control; ⁺⁺p < 0.01 tPFC transection versus control. B, Histological verification of the lesion left by a tPFC transection. Gray rectangle encompasses the anteroposterior range of all transections, and arrows point to an example of a cortical lesion trace on a midsagittal brain section (∼0.4 mm lateral to midline according to Paxinos and Watson, 1986). Shown is an illustrative depiction of the electrode placement and a typical action potential waveform of a putative 5-HT neuron (top) and a closer inspection of the lesion trace (bottom).

Figure 5.

Effect of WIN microinfused into the DR. A, Integrated firing rate histogram of a 5-HT neuron before and after intra-DR microinfusion of vehicle (0.5 μl) (n = 3 neurons). B, Integrated firing rate histogram of a 5-HT neuron before and after intra-DR microinfusion of WIN (5 μg in 0.5 μl of vehicle) showing a slight increase in single-unit firing activity immediately after infusion observed in one of four neurons. Among the other three neurons, one showed a decrease whereas the other two did not respond at all. On each histogram, 5-HT neuronal firing rate is plotted as spikes per 10 s. Horizontal bar on top represents the time course of infusion, and vertical lines at the bottom represent the frequency of neuronal burst activity such that each tick corresponds to a burst event. C, Left, An illustrative depiction of the electrode descent into the DR (shaded gray area) and the trajectory of the microcannula based on the stereotaxic atlas of Paxinos and Watson (1986). Right, Histological verification of lesions imprinted by the electrode descent (left arrow) and of the microcannula (right arrow) on a coronal brain section (∼1.2 anterior to interaural zero) showing the DR (shaded gray area). Bottom, Closer inspection of lesion traces.

Figure 6.

Effect of bilateral microinfusion of WIN into the mPFCv and latPFC on DR 5-HT neuronal activity. A, Integrated firing rate histogram of a 5-HT neuron showing a robust but slow-onset increase in single-unit activity after intra-mPFCv infusion of WIN (5 μg in 0.5 μl of vehicle) in four of five neurons. This effect was abrogated by RIM (1.0 mg/kg, i.v.). B, Integrated firing rate histogram of a 5-HT neuron before and after intra-mPFCv infusion of WIN (5 μg in 0.5 μl of vehicle) showing an abolition of increased single-unit activity resulting from total prefrontocortical transection (n = 2 neurons). The microinfusion site was anterior to the transection lesion. C, Integrated firing rate histogram of a 5-HT neuron before and after intra-mPFCv infusion of vehicle (0.5 μl), showing no apparent effect on neuronal activity (n = 4 neurons). D, Integrated firing rate histogram showing that intra-latPFC infusion of WIN (5 μg in 0.5 μl of vehicle) did not produce an increase in 5-HT single-unit activity (n = 2 neurons). On each histogram, 5-HT neuronal firing rate is plotted as spikes per 10 s. Horizontal bar represents the time course of infusion, and vertical lines at the bottom represent the frequency of neuronal burst discharge such that each tick corresponds to a burst event. E, Illustrative depiction of the placement of cannulas directed into the mPFCv (shaded region area, bregma +2.2) and the electrode descent into the dorsal raphe nucleus (interaural 0 + 1.2), based on the stereotaxic atlas of Paxinos and Watson (1986).

Figure 7.

Behavioral effects of bilateral microinfusion of WIN and RIM into the mPFCv in the rat FST. WIN (1 or 5 μg in 0.5 μl of vehicle infused for 3 min) administered 7–10 min before the FST increased total time spent swimming and decreased total time spent immobile. Bilateral microinfusion of RIM (1 μg in 0.25 μl of vehicle for 1.5 min) 1 min before microinfusion of WIN (1 μg in 0.25 μl for 1.5 min) abrogated antidepressant-like effect. RIM (1 μg in 0.5 μl of vehicle for 3 min) did not have any significant effect. n = 7–11 animals per treatment group. **p < 0.01 versus vehicle.