Faculty Bibliography

Persistent inflammatory nociception increases levels of endogenous opioids with affinity for delta opioid receptors in the ventromedial medulla and enhances the antinociceptive effects of the mu opioid receptor (MOPr) agonist [D-Ala2, N-Me-Phe4, Gly5-ol]enkephalin (DAMGO) [Hurley, R.W., Hammond, D.L., 2001. Contribution of endogenous enkephalins to the enhanced analgesic effects of supraspinal mu opioid receptor agonists after inflammatory injury. J. Neurosci. 21, 2536-2545]. It also increases levels of endogenous opioids that act at MOPr elsewhere in the CNS [Zangen, A., Herzberg, U., Vogel, Z., Yadid, G., 1998. Nociceptive stimulus induces release of endogenous beta-endorphin in the rat brain. Neuroscience 85, 659-662]. This study tested the hypothesis that a sustained release of endogenous opioids leads to a downregulation of MOPr in the locus coeruleus (LC) and induces a state of endogenous opioid tolerance. Four days after injection of complete Freund's adjuvant (CFA) in the left hindpaw of the rat, both the magnitude and duration of the antinociception produced by microinjection of DAMGO in the right LC were reduced. Saturation isotherms demonstrated a 50% decrease in MOPr B(max) in homogenates of the LC from CFA-treated rats; K(d) was unchanged. Receptor autoradiography revealed that this decrease was bilateral. The decreased efficacy of DAMGO in CFA-treated rats most likely results from a decreased number of MOPr in the LC. Microinjection of the MOPr antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) in the LC did not exacerbate hyperalgesia in the ipsilateral hindpaw or produce hyperalgesia in the contralateral hindpaw of CFA-treated rats. The downregulation in MOPr is therefore unlikely to result from the induction of endogenous opioid tolerance in the LC. These results indicate that persistent inflammatory nociception alters the antinociceptive actions of MOPr agonists in the CNS by diverse mechanisms that are nucleus specific and likely to have different physiological implications.

Despite the acknowledged advantages of studying identified populations of neurons, few studies have convincingly established that fluorescent retrograde tracers do not alter the passive membrane properties, action potential characteristics, or effects of drugs on the labeled neurons. Whole-cell patch clamp recordings were made from spinally-projecting serotonergic neurons in the rostral ventromedial medulla (RVM) and spinally-projecting noradrenergic neurons in the locus coeruleus (LC) that were retrogradely labeled with 1,1'-dioactadecyl-3,3,3',3'-tetramethylindocarbodyanine perchlorate (Dil). The passive membrane and the action potential properties of Dil-labeled (0.2%) and non-labeled serotonergic neurons in the RVM did not differ. Similarly, the passive membrane and action potential properties of non-labeled noradrenergic LC neurons did not differ from neurons labeled with 0.2% or 5% Dil. Although the mu opioid receptor agonist [D-Ala(2)-NMePhe(4)-Gly-ol(5)]enkephalin (DAMGO) produced equivalent outward currents in non-labeled noradrenergic LC neurons and those labeled with 0.2% Dil, significantly smaller currents were recorded in LC neurons labeled with 5% Dil. Baclofen, a gamma-aminobutryic acid(B) receptor agonist, also produced smaller currents in RVM neurons labeled with 5% Dil compared to 0.2% Dil. These results indicate that 0.2% Dil is suitable for retrograde labeling of brainstem neurons in vivo for subsequent in vitro electrophysiological study. However, 5% Dil is likely to confound studies of the postsynaptic actions of G-protein coupled receptor ligands.