Δ⁹-Tetrahydrocannabinol-induced desensitization of cannabinoid-mediated inhibition of synaptic transmission between hippocampal neurons in culture

Abstract

Prolonged exposure to cannabinoids results in desensitization of cannabinoid receptors. Here, we compared the desensitization produced by the partial agonist, Δ⁹-tetrahydrocannabinol (THC) to that produced by the full agonist Win55,212-2 on cannabinoid-mediated inhibition of glutamatergic synaptic transmission. Synaptic activity between rat hippocampal neurons was determined from network-driven increases in the intracellular Ca²⁺ concentration ([Ca²⁺]_i spikes). To assess the effects of prolonged treatment, cultures were incubated with cannabinoids, washed in 0.5% fatty-acid-free bovine serum albumin to ensure the removal of the lipophilic drug and then tested for inhibition of [Ca²⁺]_i spiking by Win55,212-2. In control experiments, 0.1 μM Win55,212-2 inhibited [Ca²⁺]_i spiking by 93 ± 5%. Win55,212-2 produced significantly less inhibition of [Ca²⁺]_i spiking following 18–24 h treatment with 1 μM THC (48 ± 5%) or treatment with 1 μM Win55,212-2 (29 ± 6%). Thus, THC produced significantly less functional desensitization than Win55,212-2. The desensitization produced by THC was maximal at 0.3 μM, remained stable between 1 and 7 days of preincubation and shifted the EC₅₀ of acute inhibition by Win55,212-2 from 27 to 251 nM. Differences in the long-term effects of cannabinoid receptor agonists on synaptic transmission may prove important for evaluating their therapeutic and abuse potential.

Introduction

Cannabinoids act on presynaptic CB1 receptors to inhibit glutamatergic (Shen et al., 1996) and GABAergic (Chan et al., 1998) neurotransmission. Prolonged exposure to cannabinoid agonists results in desensitization of effects mediated by CB1 receptors including activation of GTPase activity (Sim-Selley, 2003), activation of K⁺ channels (Jin et al., 1999) and inhibition of synaptic transmission (Kouznetsova et al., 2002). These cellular changes may underlie the development of tolerance to cannabinoid effects on behavior such as antinociception, hypomotility, hypothermia, and catalepsy following repeated administration (Bass and Martin, 2000, Fan et al., 1994). Desensitization of G-protein-coupled receptors is generally thought to be influenced by the intrinsic activity of the agonist (Clark et al., 1999), yet the rate of CB1 desensitization is independent of agonist efficacy (Luk et al., 2004).

Here, we studied the effects of two cannabinoid receptor agonists of different efficacies on glutamatergic activity in the synaptic network formed by rat hippocampal neurons grown in primary culture. The objective of this study was to determine whether THC, a partial agonist at CB1 receptors (Shen and Thayer, 1999), would produce less desensitization at steady state than the full agonist Win55,212-2. Differences in the long-term effects of cannabinoids on synaptic transmission may prove important for evaluating the therapeutic and abuse potential of these drugs.