Differential Agonist-Induced Desensitization of Nerve Terminal and Cell Body Mu-Opioid Receptors in Mouse Ventral Tegmental Area

Janet Lowe, Chris Bailey. University of Bath, Bath, Somerset, UK.

Agonist-induced desensitization of mu opioid receptors (MOPrs) is one of the leading mechanisms thought to underlie the development of tolerance to opioid drugs such as morphine and heroin. Although many studies have investigated desensitization of MOPrs located on cell bodies, few have examined nerve terminal MOPrs. In this study, we have investigated desensitization of MOPrs located at nerve terminals of GABAergic interneurones in the mouse ventral tegmental area (VTA); a brain region implicated in the rewarding properties of numerous drugs of abuse. We have previously demonstrated that although cell body MOPrs rapidly desensitized by more than 50% over a 10 minute agonist application, nerve terminal receptors in these neurones did not exhibit any desensitization over the same time period (Lowe and Bailey, 2010). We have further explored this differential regulation.

250 micron-thick horizontal VTA slices from 3-4 week old male C57Bl/6J mice were prepared. Evoked and miniature GABAergic inhibitory post-synaptic currents (eIPSCs or mIPSCs) were recorded in dopaminergic neurones using whole-cell patch-clamp techniques as described previously (Lowe & Bailey, 2010). For all experiments, extracellular fluid contained kynurenic acid (2 mM), strychnine (1 µM), and sulpiride (10 µM). For mIPSC recordings, tetrodotoxin (1 µM) was also included, whereas for eIPSC recordings, the g-protein inwardly rectifying potassium channel inhibitors tertiapinQ (250 nM) and barium chloride (1 mM) were included to block the effector of cell body MOPrs. In most experiments, the receptor reserve was removed by pretreating slices with the MOPr irreversible antagonist β-FNA (6 μM) for 30 minutes.

In the absence of spare receptors, MOPr agonists morphine (300 µM) and DAMGO (10 µM) inhibited the amplitude of eIPSCs by 45±9% (n = 5) and 47±5% (n = 4), respectively. The response to either agonist did not desensitize over a 10 minute application (inhibition at 10 min: morphine, 47±8%; DAMGO, 44±9%). Because activation of PKC has been shown to enhance morphine mediated desensitization in other brain regions (Bailey et al., 2004), we treated slices with the phorbol ester PMA (1 μM). However, even with PKC activation, morphine was unable to induce desensitization during a 10 minute application (peak inhibition: 26±7%, 10 min: 24±6%, n = 5). Desensitization might also be masked if receptors are rapidly internalized, recycled, and resensitized in nerve terminals. Therefore, we blocked internalization with the dynamin inhibitor dynasore (80 μM). Because the eIPSC was unstable in the presence of dynasore, we instead measured mIPSCs. In dynasore treated slices, DAMGO inhibited the frequency of mIPSCs by 32±7% (n = 5), yet the response still did not desensitize after 10 minutes of agonist treatment (30±17%).

Although nerve terminal receptors do not exhibit rapid agonist-induced desensitization, it is still possible that chronic mu opioid agonist treatments could induce MOPr desensitization. Treating slices for 7-10 hours with 30 μM Met-Enkephalin induced a dramatic reduction in nerve terminal MOPr function (post-treatment: eIPSC inhibition produced by 30 μM Met-Enkephalin was 18±9% vs control: 69±8%, n = 5-9, p = 0.003 Student’s t-test), even under conditions where the receptor reserve had not previously been removed.

These findings suggest that, in the mouse ventral tegmental area, MOPrs located at nerve terminals desensitize very slowly, requiring hours of agonist activation, whereas cell body receptors within the same neurones desensitize within minutes of agonist activation. This dramatic difference in the rate of regulation of nerve terminal and cell body MOPrs may have far reaching implications for the regulation of all G-protein coupled receptors located at nerve terminals.

Bailey CP et al. (2004) Mol. Pharm., 66, 1592-98.

Lowe JD and Bailey CP (2010) www.pa2online.org/Vol8Issue1abst108P

Funded by MRC.