Δ9-Tetrahydrocannabinol-induced desensitization of cannabinoid-mediated inhibition of synaptic transmission between hippocampal neurons in culture
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.
Section snippets
Cell culture
Rat hippocampal neurons were grown in primary culture as described previously (Wang et al., 1994) with minor modifications. Fetuses were removed on embryonic day 17 from maternal rats, anesthetized with CO2, and killed by decapitation under a protocol approved by the University of Minnesota Institutional Animal Care and Use Committee in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Hippocampi were dissected and placed in Ca2+- and Mg2+-free
Results
The effects of prolonged exposure to full and partial cannabinoid agonists were studied on excitatory synaptic transmission between rat hippocampal neurons in culture. Glutamatergic synaptic transmission was studied optically using the Ca2+-sensitive dye indo-1 AM to monitor synaptically driven increases in intracellular Ca2+ concentration ([Ca2+]i spikes). Reducing the extracellular Mg2+ concentration ([Mg2+]o) from 0.9 to 0.1 mM elicited repetitive [Ca2+]i spiking driven by glutamatergic
Discussion
Prolonged exposure to cannabinoid receptor agonists desensitized CB1-mediated inhibition of synaptic transmission between hippocampal neurons in culture. THC, an agonist of particular importance because of its recreational and therapeutic use, produced a steady state desensitization that was stable following a 7-day treatment, was maximal at concentrations over 300 nM and was smaller in magnitude than that produced by the full agonist Win 55,212-2.
The steady-state desensitization produced by THC
Acknowledgments
This work was supported by grants DA07304 and DA11806 from the National Institute on Drug Abuse (NIDA) and grant IBN0110409 from the National Science Foundation. D.L. was supported by fellowship CA106200 from the National Cancer Institute. We thank Tanner Johanns, Anthony Marsh, and Stephen Derrington for excellent technical assistance.
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