Article Figures & Data
Figures
- Fig. 1.
Exogenous opioids, but not morphine, internalize the MOR in lamina II interneurons. Shown are confocal images of lamina II MOR immunoreactivity in the L4/L5 segments of the spinal cord in animals treated with the following: A, placebo pellets;B, intrathecal endomorphin-1; C, intraplantar remifentanil; D, morphine pellets (picture1 is at 35× and picture 2 is at 105× magnification). In A and D, MOR immunoreactivity is seen predominantly on the cell membranes of neurons and their dendrites, although some smaller endosomes are visible inD. In B and C, numerous MOR immunoreactive endosomes can be seen in the cell bodies and dendrites of neurons.
- Fig. 2.
Time course of MOR recycling. The bar graph shows the average number of MOR immunoreactive endosomes found in L4/L5 lamina II neurons in rats that were injected first with 1.0 μg of intrathecal DAMGO and 10 min later with 1.0 mg/kg naloxone subcutaneously. Quantification was performed at various times after the injection of naloxone (n = 4). Also shown are confocal images of lamina II MOR immunoreactivity in the L4/L5 segments of the spinal cord at the indicated times after naloxone injection (0, 7.5, 15, 30, and 60 min). MOR internalization can be detected for up to 30 min after agonist application; note the numerous punctate inclusions in the neurons. MOR immunoreactivity has returned to the plasma membrane by 60 min. Scale bar, 20 μm.
- Fig. 3.
Opioid receptor internalization occurs in a dose-dependent manner. Slices of spinal cord were incubated in various doses of DAMGO (left) or met-enkephalin (right) for 15 min, and the average number of MOR immunoreactive endosomes per lamina II neuron was determined. Untreated control slices had 6.53 ± 1.16 endosomes per neuron for the DAMGO experiments and 9.17 ± 0.76 endosomes per neuron for the enkephalin experiments (n = 4–9 for DAMGO;n = 5 for met-enkephalin).
- Fig. 4.
Lamina II MOR internalization correlates with intrathecal DAMGO-induced analgesia in the hot plate test. Rats were injected with various doses of intrathecal DAMGO, tested for analgesia on the hot plate test, and then anesthetized and perfused for immunocytochemistry. MOR internalization was quantified in lamina II neurons from the L4/L5 segment of the spinal cord. A, Dose–response curves for changes in hot plate latency and the extent of MOR internalization. DAMGO produces spinal MOR internalization and behavioral analgesia at the same doses. Saline-injected rats had 5.58 ± 1.17 endosomes per neuron (n = 6).B, Graph of the magnitude of analgesia (hot plate latency in seconds) versus the extent of MOR internalization (number of endosomes per neuron) in lamina II of the L4/L5 segment in individual rats. Only rats that were analgesic showed significant MOR internalization. C, Confocal images of MOR immunoreactivity in lamina II from rats injected intrathecally with saline (1), 100 ng of DAMGO (2), or 1000 ng of DAMGO (3). MOR immunoreactivity is observed on the plasma membrane in vehicle-treated rats. Increasing doses of DAMGO produced increases in the amount of MOR immunoreactivity that appeared as endosomal structures in the cytoplasm.
- Fig. 5.
Noxious stimuli do not internalize the MOR. Noxious stimuli were applied to a hindlimb of an anesthetized rat. The rats were perfused 5–30 min later. Shown are confocal images of MOR immunoreactivity in lamina II of the L4/L5 segments of the spinal cord ipsilateral to the stimulus. A, This rat received a noxious pinch of the hindpaw. B, This rat received an intraplantar injection of capsaicin. C, The hindpaw of this rat was alternatively dipped and removed from 52°C water every 10 sec for 10 min (Picture 1 is at 35×, scale bar, 50 μm; picture 2 is at 105×, scale bar, 20 μm).
- Fig. 6.
Noxious stimuli do not internalize the MOR under conditions of inflammation. Shown are confocal images of MOR immunoreactivity in lamina II of the L4/L5 segments of the spinal cord ipsilateral to the inflammation and/or stimulus. A, This rat received an intraplantar injection of 5% formalin.B, This rat received a noxious pinch to a hindpaw that was inflamed after the injection of CFA, 2 d earlier.C, The hindpaw of this rat was dipped in a 52°C water bath for 2 min, 2 d after an injection of CFA (Picture1 is at 35×, scale bar, 50 μm; picture2 is at 105×, scale bar, 20 μm).
