Marijuana has been used for thousands of years as a treatment for medical conditions. However, untoward side effects limit its medical value. Here, we show that synaptic and cognitive impairments following repeated exposure to Δ 9 -tetrahydrocannabinol (Δ 9 -THC) are associated with the induction of cyclooxygenase-2 (COX-2), an inducible enzyme that converts arachidonic acid to prostanoids in the brain. COX-2 induction by Δ 9 -THC is mediated via CB1 receptor-coupled G protein βγ subunits. Pharmacological or genetic inhibition of COX-2 blocks downregulation and internalization of glutamate receptor subunits and alterations of the dendritic spine density of hippocampal neurons induced by repeated Δ 9 -THC exposures. Ablation of COX-2 also eliminates Δ 9 -THC-impaired hippocampal long-term synaptic plasticity, spatial, and fear memories. Importantly, the beneficial effects of decreasing β-amyloid plaques and neurodegeneration by Δ 9 -THC in Alzheimer’s disease animals are retained in the presence of COX-2 inhibition. These results suggest that the applicability of medical marijuana would be broadened by concurrent inhibition of COX-2.

In the present study, we unexpectedly observed that Δ-THC increases expression and activity of cyclooxygenase-2 (COX-2), an inducible enzyme that converts arachidonic acid to prostanoids both in vitro and in vivo via a CB1R-dependent mechanism. This action is opposite to the observations where the endogenous cannabinoid 2-arachidonylglycerol (2-AG) induces a CB1R-dependent suppression of COX-2 activity and expression in response to proinflammatory and excitotoxic insults (). The differential modulation of COX-2 by the exogenous cannabinoid Δ-THC and endogenous cannabinoid 2-AG appears to result from intrinsic properties of the CB1R-coupled G protein. The COX-2 induction by Δ-THC is mediated via Gβγ subunits, whereas COX-2 suppression by 2-AG is mediated through the Gαi subunit. Interestingly, the impairments in hippocampal long-term synaptic plasticity, spatial, and fear memories induced by repeated Δ-THC exposure can be occluded or attenuated by pharmacological or genetic inhibition of COX-2. Finally, the beneficial effects of reducing Aβ and neurodegeneration by Δ-THC are retained in the presence of COX-2 inhibition. Our results reveal a signaling pathway that is linked to synaptic and cognitive deficits induced by Δ-THC exposure, suggesting that Δ-THC would display its beneficial properties with fewer undesirable side effects when its COX-2 induction effect is inhibited, which may form a therapeutic intervention for medical treatments.

As it is clear now, Δ-tetrahydrocannabinol (Δ-THC) is the major psychoactive ingredient of marijuana (), and its effects are largely mediated through cannabinoid receptors (CB1R or CB2R), which are pertussis toxin (PTX)-sensitive G-protein-coupled receptors (). Previous studies demonstrate that deficits in long-term synaptic plasticity, learning, and memory by Δ-THC exposure are primarily mediated through CB1R expressed in the brain (). However, the molecular mechanisms underlying the synaptic and cognitive deficits elicited by repeated Δ-THC exposure are largely unknown.

Reduced expression of glutamate receptors and phosphorylation of CREB are responsible for in vivo Δ 9 -THC exposure-impaired hippocampal synaptic plasticity.

International Union of Basic and Clinical Pharmacology. LXXIX. Cannabinoid receptors and their ligands: beyond CB 1 and CB 2 .

Marijuana has been used for thousands of years to treat chronic pain, multiple sclerosis, cancer, seizure disorders, nausea, anorexia, and inflammatory and neurodegenerative diseases (). However, the undesirable neuropsychological and cognitive side effects greatly limit the medical use of marijuana (). The major intoxicating effects of cannabis are the impairments in synaptic and cognitive function (). These untoward effects are also the primary consequences of cannabis abuse. However, there are no currently FDA-approved effective medications for prevention and treatment of these cannabis-related disorders.

A critical issue is whether COX-2 inhibition would eliminate the beneficial effects of marijuana. To answer this question, we used 5XFAD APP transgenic mice, an animal model of Alzheimer’s disease (AD) as described previously (), to determine whether Δ-THC is capable of reducing Aβ and neurodegeneration and whether these effects are retained when COX-2 is inhibited. As shown Figures 7 A and 7B , treatment of Δ-THC once daily for 4 weeks significantly reduced the numbers of Aβ plaques and degenerated neurons in the absence and presence of Celebrex in AD animals. This information indicates that the beneficial effects of Δ-THC are preserved while COX-2 is inhibited. Meanwhile, we revealed that the reduction of Aβ by Δ-THC is not through inhibiting expression of β-site amyloid precursor protein cleaving enzyme 1 (BACE1), an enzyme responsible for synthesis of Aβ, but is likely through elevating neprilysin, an important endopeptidase that degrades Aβ ( Figure 7 C).

(B) Δ 9 -THC significantly reduces degenerated neurons detected by Fluoro-Jade C (FJC) staining in 6-month-old TG mice treated with/out Celebrex. TG mice received Δ 9 -THC (3 mg/kg) or Celebrex (1 mg/kg) once daily for 4 weeks starting at 5 months of age.

(A) Δ 9 -THC significantly reduces Aβ plaques detected using anti-4G8 antibody in 4-month-old 5XFAD APP transgenic (TG) mice in the absence and presence of COX-2 inhibition. TG mice received Δ 9 -THC (3 mg/kg) or Celebrex (1 mg/kg) once daily for 4 weeks starting at 3 months of age.

The Beneficial Effects of Reducing Aβ and Neurodegeneration by Δ 9 -THC Are Preserved in the Presence of COX-2 Inhibition

Figure 7 The Beneficial Effects of Reducing Aβ and Neurodegeneration by Δ 9 -THC Are Preserved in the Presence of COX-2 Inhibition

The Beneficial Effects of Decreasing Aβ and Neurodegeneration by Δ 9 -THC Are Preserved in the Presence of COX-2 Inhibition

Impaired long-term synaptic plasticity and memory induced by Δ-THC are largely associated with altered expression and function of glutamate receptors (). Recent evidence shows that adolescent chronic treatment with Δ-THC results in reduced density of dendritic spines and lowered length and number of dendrites in the hippocampus (). We used Thy1-GFP-expressing transgenic mice to detect morphology of dendritic spines (). As seen in Figure 5 A , repeated Δ-THC exposure significantly reduced density of dendritic spines of CA1 pyramidal neurons, especially mushroom spines in which AMPA and NMDA receptors are expressed. We found that the reduction in spines was prevented by pharmacological or genetic inhibition of COX-2. (We should mention here that the comparatively low number of mushroom-type spines in Figure 5 A may be due to the scoring criteria.) Meanwhile, Δ-THC-reduced expression of PSD-95, an important postsynaptic marker, was rescued by COX-2 inhibition ( Figure 5 B). However, Δ-THC did not alter expression of synaptophysin (Syn), a presynaptic marker. This information indicates that increased COX-2 by repeated Δ-THC exposure decreases dendritic spines and postsynaptic density. We show previously that repeated Δ-THC exposure for 7 days induces CB1R-dependent decreases in functional and surface expression of AMPA and NMDA receptor subunits (). We speculated that reduced expression of glutamate receptor subunits in the hippocampus of animals that received repeated in vivo Δ-THC exposure is likely regulated by a homeostatic mechanism. Δ-THC increased synthesis of COX-2 and its reaction product PGE, which stimulates glutamate released from presynaptic nerve terminals and astroglial cells, resulting in an extracellular accumulation of glutamate ( Figure S6 A). The increased extracellular glutamate may also result from the reduced uptake of glutamate by glutamate transporters because expression of these transporters was downregulated by repeated exposure to Δ-THC ( Figure S6 B). To this end, we used immunostaining to determine expressions of synaptic and extrasynaptic GluA1, GluN2A, and GluN2B in the hippocampal CA1 area. As shown in Figures 5 C and 5D, hippocampal expressions of both synaptic and extrasynaptic GluA1, GluN2A, and GluN2B were significantly reduced by repeated Δ-THC exposure, and the reduction was attenuated or prevented by COX-2 inhibition. This was consistent with the observations where total and surface expressions of GluA1, GluN2A, and GluN2B detected by immunoblot in WT mice were significantly decreased following exposure to Δ-THC for 7 days, but the decreases were not seen in COX-2 knockout mice ( Figure 6 ). These results indicate that reduced expression of glutamate receptor subunits and density of dendritic spines are associated with the COX-2 induction effect of Δ-THC ( Figure S7 ).

Error bars represent ± SEM;p < 0.05 andp < 0.01 compared with the vehicle control (ANOVA with Fisher’s PLSD). See also Figures S6 and S7

(C) Phosphorylation of hippocampal CREB in WT and KO mice treated with vehicle or Δ 9 -THC for 7 days (n = 3).

(B) Surface expression of GluR1, NR2A, and NR2B in WT and COX-2 KO mice treated with vehicle or Δ 9 -THC for 7 days (n = 4).

(A) Immunoblot analysis of hippocampal expression of GluR1, NR2A, and NR2B subunits in WT and COX-2 KO mice treated with vehicle or Δ 9 -THC for 7 days (n = 3).

Reduced Expression of Glutamate Receptor Subunits and Phosphorylation of CREB by Δ 9 -THC Is Rescued by COX-2 Inhibition

Figure 6 Reduced Expression of Glutamate Receptor Subunits and Phosphorylation of CREB by Δ 9 -THC Is Rescued by COX-2 Inhibition

(B) Reduced expression of glutamate transporters by Δ-THC is blocked by COX-2 inhibition, Related to Figures 5 and 6 . Hippocampal expression of glutamate transporters EAAT1, EAAT2, and EAAT3 in WT and COX-2 KO mice that received Δ-THC (10 mg/kg) and Δ-THC+Celebrex (10 mg/kg) once a day for 7 days. Immunoblot analysis was performed 24 hr after cessation of the last injection. Error bars represent ± SEM,p < 0.01 compared with the vehicle control and #p < 0.05 compared with Δ9-THC (ANOVA with Fisher’s PLSD, n = 3).

(A) Δ 9 -THC-enhanced synaptic release of glutamate is blocked by inhibition of COX-2. Hippocampal neurons in culture were treated with vehicle, Δ 9 -THC (3 μM), NS398 (10 μM), and Δ 9 -THC + NS398. Miniature spontaneous EPSCs (mEPSCs) were recorded 24 hr after treatments. Bicuculline (10 μM) and TTX (0.5 μM) were included in the external solution. Frequency and amplitude of mEPSCs were analyzed the MiniAnalysis program. Error bars present ± SEM, ∗∗ p < 0.01 compared with the vehicle control, ##p < 0.01 compared with Δ 9 -THC (n = 29 to 43 recordings, ANOVA, Bonferronni post-hoc test).

Error bars represent ± SEM; ∗∗ p < 0.01 compared with the vehicle control, # p < 0.05, and ## p < 0.01 compared with Δ 9 -THC (ANOVA with Fisher’s PLSD or Bonferronni post hoc tests).

(D) Left: enlarged immunosignals of GluA1, GluN2A, GluN2B, Syn, and their overlay. Scale bars, 1.5 μm. Right: quantification of synaptic (colocalized with Syn) and extrasynaptic (no-colocalized) GluA1, GluN2A, and GluN2B (n = 5 animals/group).

(C) Immunostaining analysis of synaptic and extrasynaptic glutamate receptor subunits. Left: schematic of a hippocampal section. The red dashed-line box marks the sampling field of immunostaining analysis. Scale bar, 200 μm. Right: representative GluA1, GluN2A, GluN2B, and Syn immunoreactivities (scale bar, 5 μm).

(B) Expression of PSD-95 and synaptophysin (Syn) in animals treated with Δ 9 -THC or NS398 for 7 days (n = 3 animals).

(A) Two-photon imaging of dendritic spines in CA1 hippocampal pyramidal neurons expressing GFP of transgenic mice. Top left: representative image of a CA1 pyramidal neurons. Scale bar, 20 μm. Top right: representative images of dendritic spine segments from animals received different treatments. Scale bars, 3 μm. Lower left: spine density in WT animals, and lower right: in COX-2 knockout (KO) mice (n = 5 animals/group).

Decreases in Dendritic Spine Density and Glutamate Receptor Expression by Δ 9 -THC Are Prevented by Inhibition of COX-2

Figure 5 Decreases in Dendritic Spine Density and Glutamate Receptor Expression by Δ 9 -THC Are Prevented by Inhibition of COX-2

Reduced expression of glutamate receptors and phosphorylation of CREB are responsible for in vivo Δ 9 -THC exposure-impaired hippocampal synaptic plasticity.

Changes in hippocampal morphology and neuroplasticity induced by adolescent THC treatment are associated with cognitive impairment in adulthood.

Reduced expression of glutamate receptors and phosphorylation of CREB are responsible for in vivo Δ 9 -THC exposure-impaired hippocampal synaptic plasticity.

Cataleptic effect and hypomotility are behavioral responses upon administering Δ-THC (). We observed that the cataleptic and locomotor depressive effects of Δ-THC were attenuated or prevented by pharmacological or genetic inhibition of COX-2 ( Figure S5 ). This means that cannabis-elicited catalepsy and locomotor depression are associated with the COX-2 induction.

(B) Ambulation (number of entries into the center area) in WT or COX-2 KO mice that received the treatments as described in (A). ∗∗ p < 0.01 compared with the vehicle control (Error bars represent ± SEM, n = 8 to12, ANOVA, Bonferronni post-hoc test).

Administration of marijuana or Δ-THC impairs learning and memory. If this impairment is associated with COX-2 induction, then inhibition of COX-2 would prevent or attenuate the deficits. To test this prediction, we determined the effect of COX-2 inhibition on spatial learning and memory using the Morris water maze test in mice that received repeated Δ-THC exposure in wild-type (WT) and COX-2 KO mice. As shown in Figures 4 B and 4C , pharmacological or genetic inhibition of COX-2 prevented Δ-THC-impaired spatial learning and memory. To further determine the role of COX-2 in Δ-THC-impaired memory, hippocampus-dependent contextual memory was determined using the fear conditioning protocol (). As seen in Figure 4 A, repeated Δ-THC exposure impaired fear memory, and this impairment was attenuated by COX-2 inhibition. These results suggest that COX-2 plays a critical role in synaptic and cognitive function deterioration consequent to repeated in vivo Δ-THC exposure ( Figure S7 ).

Error bars represent ± SEM;p < 0.01 compared with the vehicle control;p < 0.05 andp < 0.01 compared with Δ-THC (n = 9–12 animals/group, two-way ANOVA, Bonferronni post hoc test). See also Figures S5 and S7

(C) Probe trial test, which was conducted 24 hr after the cessation of the last Δ 9 -THC injection. Left: the number of times crossed the target zone. Middle: the amount of time stayed in the target quadrant. Right: swim speed in different treatments in probe trial tests.

(B) COX-2 KO and WT mice received training in the Morris water maze for 5 days without any treatments (naive). Starting at day 6, WT animals received vehicle, Δ 9 -THC (10 mg/kg), NS398 (10 mg/kg), Δ 9 -THC+NS398, once a day for 7 days. COX-2 KO mice received vehicle or Δ 9 -THC (10 mg/kg) for 7 days. Tests were performed 30 min following the injections.

(A) Impaired fear memory is attenuated by COX-2 inhibition. 24 hr after a footshock conditioning, animals were administered with Δ 9 -THC (10 mg/kg) or NS398 (10 mg/kg) once a day for 7 days. Freezing behavior was recorded 24 hr after the cessation of the last injections.

If sustained elevation of COX-2 expression and activity following repeated Δ-THC exposure contribute to impairments in long-term synaptic plasticity and cognitive function, then inhibition of COX-2 should be able to eliminate or attenuate the impairments. To test this hypothesis, we recorded hippocampal LTP in mice receiving daily injections of Δ-THC (10 mg/kg, the dosage used by other studies such as, and), NS398, Δ-THC+NS398, or vehicle for 7 consecutive days. We found that COX inhibition by NS398 rescued decreased hippocampal LTP induced by repeated in vivo exposure to Δ-THC for 7 days both at CA3-CA1 synapses ( Figure 3 A) and perforant path synapses in the dentate gyrus ( Figure S3 A). Similarly, genetic inhibition of COX-2 also prevented LTP deterioration induced by Δ-THC at both CA3-CA1 synapses ( Figure 3 B) and the perforant path ( Figure S3 B). To verify whether persistent overexpression of COX-2 impairs LTP, we recorded LTP in animals repeatedly treated with LPS, which increases COX-2. As we expected, repeated injection of LPS significantly reduced LTP, and this decrease was prevented by inhibition of COX-2 ( Figure S3 C). These data suggest that persistent elevation of COX-2 in the brain will be detrimental to integrity of synaptic structure and plasticity. Because a single dose of Δ-THC produced an increase in COX-2 expression, we wondered whether this increase alters synaptic function. To this end, we recorded long-term depression (LTD) induced by low-frequency stimulation (LFS) at hippocampal CA3-CA1 synapses and found that LTD is impaired by a single Δ-THC exposure. However, LTD is normal in COX-2 knockout animals that received a single injection of Δ-THC ( Figure S4 ). This information suggests that a single Δ-THC exposure induces a COX-2-associated impairment in LTD ().

(A) Single exposure to Δ 9 -THC impairs hippocampal LTD. LTD at CA3-CA1 synapses was induced by low-frequency stimulation (LFS, 900 stimuli at 1 Hz for 15 min). WT mice (at ages of P12 to P17) received a single injection of Δ 9 -THC (10 mg/kg, i.p.). LTD was recorded 24 hr after Δ 9 -THC injection.

(C) Repeated exposures to LPS reduce hippocampal LTP. Mice were injected with vehicle, LPS (3 mg/kg) or LPS+NS398 (10 mg/kg) once a day for 7 consecutive days. LTP was measured 24 hr after cessation of the last injection. ∗∗ p < 0.01 compared with vehicle controls. Scale bars: 0.3 mV/10 ms. Error bars represent ± SEM.

(A and B) Δ 9 -THC (10 mg/kg) was injected (i.p.) once a day for 7 days. NS398 (10 mg/kg) was injected 30 min before Δ 9 -THC injection. Perforant LTP in WT (A) and COX-2 KO (B) mice was determined 24 hr after cessation of the last injection. ∗∗ p < 0.01 compared with the vehicle control, ##p < 0.01 compared with Δ 9 -THC. (ANOVA with Bonferronni post-hoc test; n = 9 to 13 slices/6 to 9 mice).

Error bars represent ± SEM;p < 0.01 compared with vehicle controls;p < 0.01 compared with Δ-THC (ANOVA with Bonferronni post hoc test). Scale bars in (A1) and (B1), 0.3 mV/10 ms. See also Figures S3 and S4

(B) Top: representative fEPSPs recorded from COX-2 knockout (KO) mice injected with vehicle or Δ 9 -THC (10 mg/kg) once daily for 7 consecutive days. Left: time courses of changes in fEPSP slope induced by Δ 9 -THC. Right: mean values of the potentiation of fEPSPs averaged from 56 to 60 min following TBS (n = 8–12 slices/6–8 animals).

(A) Top: representative field excitatory postsynaptic potentials (fEPSPs) recorded at hippocampal CA3-CA1 synapses from WT animals repeatedly injected with vehicle, Δ 9 -THC (10 mg/kg), NS398 (10 mg/kg), or Δ 9 -THC+NS398 once daily for 7 consecutive days. LTP was measured 24 hr after cessation of the last injection. Left: time courses of changes in fEPSP slope under different treatment. Right: mean values of the potentiation of fEPSPs averaged from 56 to 60 min following TBS (n = 6– 8 slices/5–6 animals).

Reduced expression of glutamate receptors and phosphorylation of CREB are responsible for in vivo Δ 9 -THC exposure-impaired hippocampal synaptic plasticity.

To determine downstream signaling pathways of Gβγ, we detected phosphorylation of Akt, ERK, and p38MAPK by overexpression or knockdown of Gβγ in the presence and absence of Δ-THC. As shown in Figures 2 D and S2 F, Δ-THC induced phosphorylation of these signaling molecules, and the phosphorylation was inhibited by knockdown or overexpression of Gβ1γ2. Inhibition of phosphorylation of these mediators by shRNA was rescued by concurrently expressing shRNA-resistant Gβ1γ2 ( Figure 2 D). These data indicate that COX-2 induction by Δ-THC is likely through signaling of these downstream molecules of Gβγ. To further characterize this signaling pathway that regulates COX-2 expression by Δ-THC, we targeted NF-кB, which is a transcription factor regulating expression of genes, including the COX-2 gene (ptgs2). We observed that Δ-THC induced NF-кB phosphorylation in NG-108-15 cells, and this phosphorylation was inhibited by overexpression or knockdown of Gβγ and was rescued by concurrently expressing shRNA-resistant G β1γ2 ( Figures 2 E and S2 G). To determine regulation of COX-2 transcription by NF-кB, we performed a chromatin immunoprecipitation (ChIP) analysis in mixed culture of neurons and astroglial cells. As shown in Figure 2 E, a binding activity of NF-кB p65 was detected in the promoter positions (−419 to −428 bp) of ptgs2, and this interaction was enhanced by Δ-THC and inhibited by SC-514, a specific IKKβ inhibitor that inhibits p65-associated transcriptional activation of the NF-кB pathway. To further confirm the involvement of NF-кB in Δ-THC-induced increase in COX-2, COX-2 expression and NF-кB phosphorylation by Δ-THC were determined in the absence and presence of SC-514. Inhibition of IKKβ blocked Δ-THC-induced COX-2 and NF-кB phosphorylation ( Figure 2 E). Phosphorylation of Akt, ERK, p38MAPK, and NF-кB was confirmed in the hippocampus of animals that received Δ-THC ( Figure S2 H).

Because the suppression of COX-2 by 2-AG in response to proinflammatory stimuli occurs via a CB1R-dependent mechanism (), we questioned why the exogenous cannabinoid Δ-THC increases COX-2 and why the endogenous cannabinoid 2-AG suppresses COX-2 acting through the same CB1R-dependent mechanism, and we speculated that CB1R may not be the key molecule responsible for differential regulation of COX-2 expression upon exposure to cannabinoids. CB1R is coupled to a PTX-sensitive Gi/o protein, and activation of CB1R releases Gβγ subunits from the GTP-bound Gαi subunit (). Earlier studies show that activation of CB1R is capable of inducing Gβγ-mediated response (). We hypothesized that Gβγ and Gαi may differentially mediate COX-2 induction or suppression by exogenous Δ-THC or endogenous 2-AG. To test this prediction, we first overexpressed Gβγ subunits by transfection with plasmids carrying β1 and γ2 subunits in NG108-15 cells, which express native CB1R ( Figures S2 A and S2B). Whereas Δ-THC still increased expression of COX-2 mRNA in culture transfected with the control vector, it did not increase COX-2 in culture overexpressing β1 and γ2 subunits ( Figure 2 A). In subsequent experiments, β1 and γ2 subunits were silenced by small hairpin RNA (shRNA). Knockdown of β1γ2 by shRNA suppressing endogenous β1γ2 also blocked COX-2 induction by Δ-THC in NG108-15 cells, and the blockade was rescued by concurrently expressing shRNA-resistant β1γ2 ( Figures 2 A and S2 E). This indicates that COX-2 induction by Δ-THC is likely mediated through Gβγ. To further confirm that Gβγ mediates COX-2 induction by Δ-THC, we treated mixed culture of hippocampal neurons and astroglial cells (∼5%–10%) with a membrane-permeable Gβγ-binding peptide mSIRK to disrupt the function of Gβγ (). As a negative control, we used a variant mSIRK with a point mutation of Leuto Ala (LA-mSIRK). As shown in Figure 2 B, disruption of Gβγ activity by mSIRK also blocked COX-2 induction by Δ-THC, whereas it failed to block the suppression of COX-2 by 2-AG in response to LPS, a commonly used COX-2 inducer (). PTX treatment also blocked Δ-THC-induced increase in COX-2. Interestingly, application of 2-AG failed to suppress Δ-THC-induced increase in COX-2 ( Figures 2 B and S2 I). To test the prediction that Gαi mediates COX-2 suppressive effect by 2-AG, we silenced Gαi using a lentiviral vector in mixed culture of neurons and astroglial cells ( Figure S2 C). As illustrated in Figures 2 C and S2 D, silencing Gαi1, but not Gαi2 or Gαi3, blocked the suppression of COX-2 by 2-AG in response to the LPS stimulus, and this blocking effect was rescued by concurrently expressing shRNA-resistant Gαi1 ( Figures 2 C and S2 E). Knockdown of Gαi1, Gαi2, or Gαi3 did not block COX-2 induction by Δ-THC ( Figures 2 C and S2 D). These results indicate that COX-2 induction by Δ-THC is likely mediated via Gβγ, whereas COX-2 suppression by 2-AG is likely mediated through Gαi1 ( Figure S7 ).

(E) Left: Δ 9 -THC induces phosphorylation of NF-кB, and the effect is blocked by Gβ1γ2 shRNA in NG108-15 cells. Middle: binding of NF-кB p65 in the promoter region of the COX-2 gene (ptgs2) by ChIP analysis. Right: Δ 9 -THC-induced NF-кB phosphorylation and COX-2 expression are blocked by IKKβ inhibition in mixed culture of neurons and astroglial cells.

(D) Δ 9 -THC induces phosphorylation of Akt, ERK, and p38MAPK. The phosphorylation is inhibited by knockdown of Gβγ2, and the inhibition is rescued by expressing shRNA-resistant Gβ1γ2.

(C) Silencing the Gαi1 subunit blocks 2-AG-suppressed COX-2 but does not affect the elevation of COX-2 by Δ 9 -THC in mixed culture of neurons and astroglial cells treated with the lentiviral vector expressing Gαi1 shRNA or shRNA-resistant Gαi1.

(B) Disruption of Gβγ subunits blocks Δ 9 -THC-elevated COX-2 but does not prevent suppression of COX-2 by 2-AG in response to LPS stimulus in mixed culture of hippocampal neurons and astroglial cells (∼10%). The culture was treated with a membrane-permeable Gβγ-binding peptide mSIRK or a single point mutated (Leu 9 to Ala) Gβγ-binding peptide mSIRK (L 9A -mSIRK) in the absence and presence of Δ 9 -THC, LPS, PTX, 2-AG.

(A) Overexpression or knockdown of β1 and γ2 subunits eliminates Δ 9 -THC-increased COX-2 mRNA detected by qPCR in NG108-15 cells. Error bars represent ± SEM; ∗∗ p < 0.01 compared with the vehicle control (ANOVA, Fisher’s PLSD, n = 6). NG108-15 cells were transfected with pcDNA3.1 plasmids encoding Gβ 1 and Gγ 2 subunits, the pLL3.7 vector expressing Gβ1 and Gγ2 shRNA, or the vector expressing shRNA-resistant Gβ1γ2 in the absence and presence of Δ 9 -THC.

(I) 2-AG does not inhibit Δ 9 -THC-induced increase in COX-2 expression in mixed culture of neurons and astroglial cells.

(F) Δ 9 -THC (30 μM)-induced phosphorylation of Akt, ERK and p38MAPK is inhibited by overexpression of Gβ1γ2 in NG108-15 cells.

(E) Left: immunoblot analysis of Gβ1; middle: Gγ2 levels in NG108-15 cells; right: Gαi1 in mixed culture of hippocampal neurons and astroglial cells transduced with vectors or lentivirus expressing scramble, Gβ1-, Gγ2-, and Gαi1-shRNA, and shRNA-resistant Gβ1, Gγ2, and Gαi1 ().

(D) Silencing the Gαi1 shRNA blocks 2-AG-induced suppression of COX-2 induced by LPS, while expression of Gαi2 or Gαi3 shRNA fails to blocks the COX-2 suppressive effect by 2-AG. Expression of Gαi1, Gαi2, or Gαi3 did not affect the increase in COX-2 by Δ 9 -THC in mixed culture of hippocampal neurons and astroglial cells.

(C) Expression of Gαi1, Gαi2, and Gαi3 in mixed neuronal and astroglial cell culture treated with lentivrus expressing individual Gαi1 (1), Gαi2 (2), and Gαi3 (3) shRNAs.

(A and B) Overexpression and knockdown of Gβ1γ2 in NG108-15 cells. Expression Gβ1 and Gγ2 was detected using qPCR analysis (Error bars represent ± SEM, n = 3 to 6).

International Union of Basic and Clinical Pharmacology. LXXIX. Cannabinoid receptors and their ligands: beyond CB 1 and CB 2 .

Because undesirable side effects elicited by cannabinoids are primarily mediated by CB1R (), we wondered whether COX-2 induction by Δ-THC is mediated via CB1R. As shown in Figures 1 E and1F, Δ-THC-induced increase in COX-2 in the hippocampus was blocked either by Rimonabant (RIM), a selective CB1R antagonist, or by genetic deletion of CB1R. To determine whether the increase in COX-2 by Δ-THC occurs in neurons or astroglial cells, we made different conditions in cultures as described previously (). We found that, although Δ-THC induced a CB1R-dependent increase in COX-2 expression both in neuronal and astroglial cell-enriched cultures, the increase was more pronounced in astroglial cell-enriched cultures than in neuronal culture ( Figure 1 G). Our data provide convincing evidence that COX-2 induction by Δ-THC both in vivo and in vitro is mediated via CB1R.

Identification of CBRs led to discovery of several endogenous cannabinoids, including anandamide (AEA) and 2-arachidonylglycerol (2-AG), which are the most-studied endocannabinoids involved in a variety of physiological, pharmacological, and pathological processes (). 2-AG, the most abundant endocannabinoid, plays significant roles in synaptic modification, resolution of neuroinflammation, and neuronal survival (). In particular, its anti-inflammatory and neuroprotective effects in response to proinflammatory and neurotoxic insults appear to be through limiting COX-2 signaling (). Because acute inhibition of COX-2 by selective COX-2 inhibitors has been shown to decrease hippocampal long-term potentiation (LTP) and impairs memory consolidation (), we thus wondered whether impairments of synaptic plasticity and memory by marijuana result from a COX-2 suppressive effect. To assess this, we first analyzed hippocampal expression and activity of COX-2 in mice that received Δ-THC. Unexpectedly, in vivo exposure to Δ-THC produced a dose- and time-dependent induction of COX-2 in the brain, rather than suppression ( Figures 1 A and 1B ), whereas expression of COX-1 was unaffected by Δ-THC ( Figure S1 A available online). The increase in COX-2 expression induced by Δ-THC was accompanied by elevated production of prostaglandin E(PGE), which could be inhibited by the selective COX-2 inhibitor Celebrex or genetic inhibition of COX-2 ( Figures 1 C and S1 B). To confirm the ability of exogenous cannabinoids to induce COX-2, we assessed COX-2 expression and PGEproduction in animals injected with the synthetic cannabinoid CP55,940 (CP). As expected, CP produced more pronounced effects on COX-2 expression and PGEsynthesis ( Figures S1 C–S1E). The increase in PGEcould be blocked by NS398, another selective COX-2 inhibitor. In addition, we observed that COX-2 expression was steadily elevated in animals injected with Δ-THC once daily for 7 consecutive days, although the magnitude of increase in COX-2 was not as intensified as that of a single injection ( Figure 1 D). This indicates that expression of COX-2 is persistently elevated upon repeated exposure to Δ-THC ( Figure S7 ).

All the data are presented as mean ± SEM, ∗∗ p < 0.01 compared with the vehicle control, ##p < 0.01 compared with Δ 9 -THC or CP (one-way ANOVA with Fisher’s PLSD).

(C–E) Synthetic cannabinoid CP55,940 (CP) increases COX-2 expression and PGE2 synthesis. COX-2 expression and PGE2 were detected 4 hr after CP (10 mg/kg). NS398 (10 mg/kg) was administered 30 min prior to injection of CP. Hippocampal COX-2 mRNA was detected using the qPCR analysis. Increase in PGE2 by CP is inhibited by NS398 (n = 5 to 7/group).

(B) Δ 9 -THC increase hippocampal PGE2 synthesis and the increase is blocked by Celebrex (Celeb, 10 mg/kg, n = 10 animals/group)). PGE2 was detected 30 min after injection of Δ 9 -THC.

(A) Δ 9 -THC does not increase in COX-1 expression in the hippocampus. COX-1 protein was detected 4 hr after Δ 9 -THC injection (10 mg/kg, n = 3).

All the data are presented as mean ± SEM;p < 0.05,p < 0.01 compared with the vehicle controls, #p < 0.05, andp < 0.01 compared with Δ-THC (one-way ANOVA, Fisher’s PLSD). See also Figures S1 and S7

(G) Δ 9 -THC increases COX-2 both in neurons and astroglial cells in culture, and the increase is blocked by RIM. COX-2 was assayed 12 hr after treatments (n = 6).

(E) COX-2 induction by Δ 9 -THC (10 mg/kg) is blocked by Rimonabant (RIM, 5 mg/kg). Hippocampal COX-2 was detected 4 hr after Δ 9 -THC injection (n = 3). RIM was injected 30 min prior to Δ 9 -THC injection.

(D) COX-2 is persistently elevated in animals that received repeated injections of Δ 9 -THC (10 mg/kg, i.p.) once a day for 7 consecutive days. COX-2 was analyzed 24 hr after secession of the last injection (n = 3).

(C) Δ 9 -THC increases synthesis of PGE 2 , and the increase is blocked by Celebrex (Celeb) or genetic inhibition of COX-2 (COX-2 knockout). PGE 2 was detected 4 hr after Δ 9 -THC injection (10 mg/kg). Celebrex (10 mg/kg) was injected 30 min prior to Δ 9 -THC injection (n = 10/group).

COX-2, but not COX-1, activity is necessary for the induction of perforant path long-term potentiation and spatial learning in vivo.

International Union of Basic and Clinical Pharmacology. LXXIX. Cannabinoid receptors and their ligands: beyond CB 1 and CB 2 .

Discussion

The results presented here demonstrate that impaired synaptic and cognitive function induced by repeated Δ9-THC exposure is associated with a previously unrevealed CB1R-Gβγ-Akt-ERK/MAPK-NF-кB-COX-2 signaling pathway. It has been long known that use of marijuana induces neuropsychiatric and cognitive deficits, which greatly limit medical use of marijuana. Synaptic and memory impairments are also the consequence of cannabis abuse. However, the molecular mechanisms underlying undesirable effects by cannabis are largely unknown. We discovered in this study that pharmacological or genetic inhibition of COX-2 eliminates or attenuates synaptic and memory impairments elicited by repeated Δ9-THC exposure, suggesting that these major adverse effects of cannabis on synaptic and cognitive function can be eliminated by COX-2 inhibition, which would broaden the use of medical marijuana.