Desensitization of Cannabinoid-Mediated Presynaptic Inhibition of Neurotransmission Between Rat Hippocampal Neurons in Culture

Abstract

Prolonged exposure to cannabinoids results in tolerance in vivo and desensitization of cannabinoid receptors in vitro. We show here that cannabinoid-induced presynaptic inhibition of glutamatergic neurotransmission desensitized after prolonged exposure to the cannabinoid receptor agonist ( Win55,. Synaptic activity between hippocampal neurons in culture was determined from network-driven increases in intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i spikes) and excitatory postsynaptic currents. Win55,212-2-induced (100 nM) inhibition partially desensitized after 2 h and completely desensitized after 18-to 24-h exposure. The desensitization could be overcome by higher concentrations of agonist as indicated by a parallel rightward shift of the concentration response curve from an EC 50 of 2.7 Ϯ 0.3 nM to 320 Ϯ 147 nM for inhibition of [Ca 2ϩ ] i spiking and from 43 Ϯ 17 nM to 4505 Ϯ 403 nM for inhibition of synaptic currents, suggesting that this phenomenon may underlie tolerance. Presynaptic expression of dominant negative G-protein-coupled-receptor kinase (GRK2-Lys220Arg) or ␤-arrestin (319 -418) reduced the desensitization produced by 18-to 24-h pretreatment with 100 nM, Win55,212-2 suggesting that desensitization followed the prototypical pathway for G-protein-coupled receptors. Prolonged treatment with Win55,212-2 produced a modest increase in the EC 50 for adenosine inhibition of synaptic transmission and pretreatment with cyclopentyladenosine produced a slight increase in the EC 50 for Win55,212-2, suggesting a reciprocal ability to produce heterologous desensitization. The long-term changes in synaptic function that accompany chronic cannabinoid exposure will be an important factor in evaluating the therapeutic potential of these drugs and will provide insight into the role of the endocannabinoid system.

Differential β-arrestin2 requirements for constitutive and agonist-induced internalization of the CB1 cannabinoid receptor

“…As described in the introduction, the binding between β-arr2 and CB 1 R as well as a role for β-arrestins in the desensitization of CB 1 R have been demonstrated by former studies (Bakshi et al, 2007;Daigle et al, 2008aDaigle et al, , 2008bJin et al, 1999;Kouznetsova et al, 2002;Nguyen et al, 2012;Singh et al, 2011). Therefore, the role of β-arr2 in CB 1 R endocytosis may seem to be a logical consequence of its recruitment to the receptor.…”

“…Numerous studies suggest that β-arr2 is involved in the regulation of CB 1 R. Coexpression of both GRK3 and β-arr2 was needed for the proper desensitization of the receptor in Xenopus oocyte (Jin et al, 1999), and dominant negative GRK2 and β-arrestin constructs reduced CB 1 R desensitization in hippocampal neurons (Kouznetsova et al, 2002). In β-arr2 knockout mice desensitization and downregulation of CB 1 R were impaired in certain regions of the central nervous system (Nguyen et al, 2012).…”

Rapid CB1 cannabinoid receptor desensitization defines the time course of ERK1/2 MAP kinase signaling

“…The physical association of βarrestin with the activated receptor has been shown to uncouple many GPCR's from their cognate Gproteins, inducing homologous desensitization (Gainetdinov et al, 2004). In Xenopus oocytes and cultured hippocampal neurons, homologous CB 1 receptor desensitization is mediated in part by βarrestin-2 (Jin et al, 1999;Kouznetsova et al, 2002). In these studies βarrestin overexpression or dominant negative βarrestin-2 mutants were used to reveal the necessity of this protein for the desensitization of CB 1 receptor-mediated activation of GIRK channels or inhibition of voltage-gated Ca 2+ channels.…”

“…βarrestin-2 has been implicated as a key regulator of desensitization of CB 1 receptor-mediated coupling to GIRK currents and excitatory glutamatergic transmission in Xenopus oocytes and cultured hippocampal neurons, respectively (Jin et al, 1999;Kouznetsova et al, 2002). These studies determined the necessity of βarrestin-2 for GPCR desensitization using dominant negative arrestin constructs or direct protein over-expression.…”

“…The physical interaction of βarrestin with the phosphorylated receptor reduces the affinity of the GPCR for G-proteins, likely via a mechanism involving steric hindrance, thereby attenuating signaling (Dewire et al, 2007). Previous studies have found that the βarrestin-2 isoform of βarrestin and GRK3 are capable of desensitizing CB 1 receptor-mediated activation of GIRK channels and inhibition of neurotransmission (Jin et al, 1999;Kouznetsova et al, 2002). Furthermore, serines 426 and 430 in the proximal carboxy-terminus of CB 1 are the likely GRK3 phosphorylation sites underlying βarrestin-2 mediated CB 1 receptor desensitization of GIRK activation (Jin et al, 1999).…”