Δ⁹-Tetrahydrocannabinol antagonizes endocannabinoid modulation of synaptic transmission between hippocampal neurons in culture

Abstract

Cannabinoids inhibit excitatory synaptic transmission between hippocampal neurons in culture. Δ⁹-tetrahydrocannabinol (THC), the principal psychoactive component in marijuana, acts as a partial agonist at these synapses. Thus, THC inhibited but did not block synaptic transmission when applied alone and, when applied in combination with WIN552212-2, it partially reversed the effects of this full agonist. Here, we address the question of how THC might interact with endocannabinoid signaling. Reducing the extracellular Mg²⁺ concentration to 0.1 mM elicited a repetitive pattern of glutamatergic synaptic activity that produced intracellular Ca²⁺ concentration spikes that were measured by indo-1-based microfluorimetry. The endocannabinoid, 2-arachidonyl glycerol (2-AG) produced a concentration-dependent and complete inhibition of spike frequency with an EC₅₀ of 63±13 nM. 2-AG (1 μM) inhibition of spiking was blocked by SR141716A (1 μM). THC (100 nM) antagonized the actions of 2-AG producing a parallel shift in the concentration–response relationship for 2-AG (EC₅₀ of 1430±254 nM). The attenuation of 2-AG (1 μM) inhibition of synaptic activity by THC was concentration-dependent with an IC₅₀ of 42±9 nM. These results demonstrate that THC can antagonize endocannabinoid signaling. Thus, the effects of THC on synaptic transmission are predicted to depend on the level of endocannabinoid tone.

Introduction

Cannabis sativa has been used recreationally and therapeutically for thousands of years (Mechoulam and Hanus, 2000). Marijuana is the most commonly used illicit drug in the United States (Abood and Martin, 1992). Medicinal applications for Δ⁹-tetrahydrocannabinol (THC), the principal psychoactive ingredient in marijuana, focus on its analgesic, antispasmodic, appetite stimulant, and antiemetic properties (Pertwee, 2000). THC exerts its effects by interacting with receptors that are part of an endogenous cannabinoid signaling system (Piomelli et al., 1998).

Cannabinoids act on G-protein-coupled receptors primarily located in the brain (CB1) and the immune system (CB2) (Howlett, 1998). These receptors couple via inhibitory G-proteins to activate K⁺ channels and members of the MAP kinase family and to inhibit adenylyl cyclase and voltage-gated Ca²⁺ channels (Porter and Felder, 2001). Several endogenous ligands for these receptors have been identified including arachidonyl ethanolamide (anandamide), 2-AG and noladin ether (Fowler, 2003). The best-characterized integrative model for how this system modulates neuronal activity is depolarization-induced suppression of excitatory (DSE) (Kreitzer and Regehr, 2001a) or inhibitory (DSI) neurotransmission (Wilson and Nicoll, 2001). A strong postsynaptic depolarization elevates the intracellular Ca²⁺ concentration ([Ca²⁺]_i) stimulating the production of endocannabinoids, which diffuse across the synapse in a retrograde direction to act on presynaptic CB1 receptors that then inhibit neurotransmitter release. Generally, cannabinoids acting on CB1 receptors are thought to mimic the endogenous ligands (Shen et al., 1996). However, some cannabinoids are partial agonists making it less clear how endocannabinoids and exogenous cannabinoids in combination will affect synaptic transmission (Mackie et al., 1993, Pan et al., 1996).

THC is a partial agonist with a low efficacy for stimulation of [S³⁵]GTPγS binding (Sim et al., 1996) and inhibition of synaptic transmission (Shen and Thayer, 1999). The functional response to a partial agonist, because of the low intrinsic activity for receptor activation, is especially sensitive to receptor density. Furthermore, the level of endocannabinoid tone will determine whether it acts as an agonist or antagonist.

Here, we test the hypothesis that THC can antagonize endocannabinoid-mediated inhibition of excitatory synaptic transmission. Using [Ca²⁺]_i spiking as an index of glutamatergic synaptic transmission, we found that THC attenuated the inhibition of synaptic transmission produced by the endocannabinoid 2-AG. These results suggest that THC might exert effects on neurotransmission that are synapse specific and dependent on the activation state of the endocannabinoid system. A preliminary report of this work has appeared (Kelley and Thayer, 2003).