Hippocampal neurotoxicity of Delta9-tetrahydrocannabinol

Abstract

Marijuana consumption elicits diverse physiological and psychological effects in humans, including memory loss. Here we report that Delta9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, is toxic for hippocampal neurons. Treatment of cultured neurons or hippocampal slices with THC caused shrinkage of neuronal cell bodies and nuclei as well as genomic DNA strand breaks, hallmarks of neuronal apoptosis. Neuron death induced by THC was inhibited by nonsteroidal anti-inflammatory drugs, including indomethacin and aspirin, as well as vitamin E and other antioxidants. Furthermore, treatment of neurons with THC stimulated a significant increase in the release of arachidonic acid. We hypothesize that THC neurotoxicity is attributable to activation of the prostanoid synthesis pathway and generation of free radicals by cyclooxygenase. These data suggest that some of the memory deficits caused by cannabinoids may be caused by THC neurotoxicity.

Fig. 1.

THC is toxic for cultured hippocampal neurons. Primary hippocampal neurons were treated with various concentrations of THC at high doses (A) (in μm):open squares, 3.5; open triangles, 5.0;filled circles, 7.5; filled triangles, 10; and filled squares, 20; or low doses (B) (in μm): open squares, 0.20; open triangles, 0.38;filled circles, 0.50; filled triangles, 1.0; and filled squares, 2.0. C, Primary hippocampal neurons were treated with 3.5 μm THC for the indicated periods (15, 30, or 45 min or 20 hr), followed by removal of the drug and reincubation in conditioned media overnight. Cell viability was measured by the MIT assay. One hundred percent viability is defined as the MTT conversion by cells treated with the carrier for the duration of the experiment.

Fig. 2.

THC toxicity requires activation of the CB1 receptor. Neurons were treated with 10 μm THC in the presence or absence of the CB1 antagonist SR141716A (SR). Cell viability was analyzed 5 hr after treatment by the MTT assay. Neurons were preincubated with SR for 20 min before treating with THC.

Fig. 3.

THC toxicity is not attributable to changes in cAMP. Hippocampal neuron viability was measured after treatment with 10 μm THC alone (THC), THC with 200 ng/ml pertussis toxin (Ptx; pretreated for 16 hr), with THC in the presence of 10 μm forskolin (Fsk), or with THC in the presence of 2 μg/ml actinomycin D (Act D). With the exception of Ptx, neurons were treated with reagents for 5 hr. p values are from Student’st test: NS, not significant; **p < 0.005. Data are triplicates from one representative experiment. Comparable results were obtained in four independent experiments. Ctrl, Control treatment with 0.01% ethanol, the carrier for THC. Error bars indicate ±SD.

Fig. 4.

THC induces a delayed increase in intracellular Ca²⁺ that is dependent on extracellular Ca²⁺. [Ca²⁺]_i of THC-treated primary hippocampal neurons was monitored by fura-2 fluorescent imaging. The calibrated [Ca²⁺]_i (nm) was plotted against time (minutes). A, THC was added to neurons in the presence of 1.5 mm CaCl₂ (THC + Ca²⁺) at the indicated time (arrow). No increase in intracellular Ca²⁺ was seen when the ethanol carrier (0.01%) was added alone (data not shown) or when THC was added to cells incubated without Ca²⁺ in the presence of 5 mm EGTA (THC − Ca²⁺). Four mm LaCl₃(B) or 5 μm SR141716A (C) blocked increases in [Ca²⁺]_i caused by 10 μmTHC.

Fig. 5.

THC neurotoxicity is blocked by inhibitors of the PLA₂ cyclooxygenase pathway and antioxidants, but not by Ca²⁺ chelation. Neurons were treated with 0.2% ethanol (Ctrl), 3.5 μm THC (THC), or THC in the presence of 0.2 μmquinacrine (Quin), 1 μm indomethacin (Indo), 5 μm aspirin (Asp), 5 μm NDGA, 1.5 mm EGTA, or 20 μm α-tocopherol (Vit E). After 20 hr, neurons were assayed for viability as described in Materials and Methods. Neurons were preincubated with inhibitors for 60 min before adding THC. Numbers in parentheses represent the number of independent experiments, each performed in triplicate. Error bars indicate SD.p values are from Student’s t test. *p < 0.05; **p < 0.0005; NS, not significant.

Fig. 6.

THC induces arachidonic acid release from hippocampal neurons. Arachidonic acid release was monitored in cultured hippocampal neurons after treatment with THC. Hippocampal neurons were maintained in culture for 7–8 d and loaded with [³H]arachidonic acid for 3–4 hr and treated as described in Materials and Methods with 3.5 μm(A) or 10 μm(B) THC. SR141716A (SR) pretreatment (5 μm) was done 5 min before THC treatment. Pertussis toxin (Ptx) was added at 300 ng/ml, 12 hr before the addition of THC. Error bars indicate SD.

Fig. 7.

THC induces nuclear shrinkage and genomic DNA breakage in primary hippocampal neurons. Neurons were maintained for 10 d before treatment with 0.01% ethanol for 3 d (A, D), 2.0 μm THC for 3 d (B, E), or 10 μmTHC for 6 hr (C, F). Cells were analyzed using the TUNEL assay (A–C) coupled to horseradish peroxidase assay or stained with 2.5 μg/ml of Hoechst 33342 (D–F) as described in Materials and Methods. Light microscopic images of horseradish peroxidase staining and counter staining with 0.1% toluidine blue (A–C) and fluorescent images (D–F) are shown. Thin arrows indicate representative examples of condensed chromatin;thick arrows indicate normal chromatin. Scale bars, 50 μm.

Fig. 8.

THC induces nuclear shrinkage and DNA cleavage in neurons of hippocampal slices. Hippocampal slices perfused with 0.01% ethanol, the carrier for THC, (A, C,E, G) or with 2.5 μm THC (B, D, F, H) for 2 hr followed by 3 hr of perfusion in buffer alone. Slices were stained with propidium iodide (A–D) or subjected to TUNEL assays (E–H) as described in Materials and Methods. Confocal microscopic images of the CA1 pyramidal cell layer are shown. s.o., Stratum oriens; m., CA1 pyramidal cell layer;s.r., stratum radiatum of the hippocampus. Scale bars: A, B, 50 μm;C, D, 10 μm; E,F, 100 μm; and G, H, 25 μm.

Fig. 9.

Signaling pathways contributing to THC-induced neuronal death. It is hypothesized that excessive stimulation of cannabinoid receptors (CB1) by THC stimulates the production of arachidonic acid (AA) by several pathways. Cyclooxygenase (COX) catalyzes the formation of prostaglandins (PGs), thromboxanes (TXs), and reactive oxygen species (ROS), which stimulates peroxidation of lipids, proteins, and DNA. In addition, cannabinoid-induced transcriptional events may also contribute to THC induction of neuron death.