Assessing Opioid Tolerance Mechanisms in an Isolated Murine Dorsal Root Ganglia Neuron Model

Our current understanding of tolerance development to opioids has largely been collected through whole‐animal studies and cell culture models, yet many questions related to the underlying mechanisms remain. One approach our lab has employed is the use of isolated dorsal root ganglia (DRG) neurons from adult mice as an ex vivo native cell culture model to study the effects of opioids on neurons and to further our understanding of tolerance development within a single cell. Our initial studies focused on comparing morphine and oxycodone, two opioids widely used clinically for the treatment of pain. Interestingly, oxycodone and morphine are known to differ in antinociceptive potency and oral bioavailability when tested in whole animals, yet we found using whole‐cell patch clamp electrophysiology that acute oxycodone and morphine had similar potencies in DRG neurons. A significant decrease in neuronal excitability as measured by an increase in the potential required to fire an action potential (i.e. threshold potential) from baseline was observed following the application of 3 μM oxycodone (p < 0.01) or morphine (p < 0.05). Marked increases in threshold potential occurred rapidly, within five to ten minutes of drug application. When incubated in media containing 3 μM morphine, DRG neurons displayed tolerance development to a morphine challenge two days later as indicated by no change in threshold potential (p < 0.05). Tolerance was observed following overnight incubation in media containing 10 μM of either morphine or oxycodone (p < 0.05). β‐arrestin 2 knockout (β‐arr2 KO) mice do not develop antinociceptive tolerance to morphine in vivo, and we found that DRG neurons isolated from β‐arr2 KO mice and incubated in 10 μM morphine continued to respond to a 3 μM morphine challenge, which showed that tolerance to morphine did not develop in a single‐cell preparation (p < 0.05). We further observed that overnight oxycodone incubation did not produce tolerance in β‐arr2 KO DRGs (p < 0.05), providing evidence that β‐arrestin 2 plays a role in tolerance development to both oxycodone and morphine. Our lab has also shown a substantial role of protein kinase C (PKC) in morphine antinociceptive tolerance in vivo using warm water tail withdrawal studies. We tested an inhibitor of PKC, Bisindolylmaleimide XI, on DRG neurons incubated overnight with 10 μM oxycodone and found a rapid reversal of tolerance to oxycodone (p < 0.01). These data indicate that PKC‐mediated mechanisms also underlie oxycodone tolerance, similarly to that of morphine tolerance, and operate at the single‐cell level. Opioid tolerance mechanisms are undoubtedly complicated and involve multiple signaling molecules, including β‐arrestin 2 and PKC. DRG neurons are a solid model bridging in vivo and in vitro studies to investigate cellular mechanisms of opioid tolerance. Support or Funding Information 5T32DA007027P30DA033934 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .