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. 2013 Jul;38(8):1472-84.
doi: 10.1038/npp.2013.46. Epub 2013 Feb 12.

Pain after discontinuation of morphine treatment is associated with synaptic increase of GluA4-containing AMPAR in the dorsal horn of the spinal cord

David Cabañero  1 , Alyssa Baker, Shengtai Zhou, Gregory L Hargett, Takeshi Irie, Yan Xia, Hélène Beaudry, Louis Gendron, Zara Melyan, Susan M Carlton, Jose A Morón
Affiliations

Pain after discontinuation of morphine treatment is associated with synaptic increase of GluA4-containing AMPAR in the dorsal horn of the spinal cord

David Cabañero et al. Neuropsychopharmacology. 2013 Jul.

Abstract

Withdrawal from prescribed opioids results in increased pain sensitivity, which prolongs the treatment. This pain sensitivity is attributed to neuroplastic changes that converge at the spinal cord dorsal horn. We have recently reported that repeated morphine administration triggers an insertion of GluA2-lacking (Ca(2+)-permeable) α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPAR) in the hippocampus. This finding together with the reported involvement of AMPAR in the mechanisms underlying inflammatory pain led us to hypothesize a role for spinal AMPAR in opioid-induced pain behavior. Mice treated with escalating doses of morphine showed hypersensitivity to mechanical stimulation. Intrathecal administration of a Ca(2+)-permeable AMPAR selective blocker disrupted morphine-induced mechanical sensitivity. Analysis of the expression and phosphorylation levels of AMPAR subunits (GluA1/2/3/4) in homogenates and in postsynaptic density fractions from spinal cord dorsal horns showed an increase in GluA4 expression and phosphorylation in the postsynaptic density after morphine. Co-immunoprecipitation analyses suggested an increase in GluA4 homomers (Ca(2+)-permeable AMPAR) and immunohistochemical staining localized the increase in GluA4 levels in laminae III-V. The excitatory postsynaptic currents (EPSCs) recorded in laminae III-V showed enhanced sensitivity to Ca(2+)-permeable AMPAR blockers in morphine-treated mice. Furthermore, current-voltage relationships of AMPAR-mediated EPSCs showed that rectification index (an indicator of Ca(2+)-permeable AMPAR contribution) is increased in morphine-treated but not in saline-treated mice. These effects could be reversed by infusion of GluA4 antibody through patch pipette. This is the first direct evidence for a role of GluA4-containing AMPAR in morphine-induced pain and highlights spinal GluA4-containing AMPAR as targets to prevent the morphine-induced pain sensitivity.

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Figures

Figure 1
Figure 1
Repeated morphine induces pain sensitivity to mechanical stimuli. Measurements are taken 12 h after repeated saline (□) or morphine administration (▪). Values are expressed as mean %±SEM from baseline, set to 100 (a, b, d), except for the hot plate test, which shows raw data (c). (a) Mechanical paw thresholds measured with von Frey test, n=6. (b) Paw withdrawal latencies to heat stimulation, Hargreaves test, n=6.(c) Paw withdrawal latencies to heat stimulation, hot plate test, n=6–7. (d) tail-flick latencies to heat stimulation, hot tail immersion test, n=6–7. *P<0.05, saline vs morphine; unpaired t-test. #P<0.05, vs baseline, paired t-test.
Figure 2
Figure 2
Intrathecal administration of naspm reverses mechanical hypersensitivity in morphine-treated mice. Twelve hours after repeated morphine or saline administration, mice are injected i.t. with PBS or naspm and evaluated 60 min later. (a) Dose–response relationship for naspm in morphine-treated mice. The PBS group was significantly different from baseline but all naspm groups were not; #p<0.05, vs baseline, paired t-test. (b) 0.01 nmol of naspm reversed mechanical hypersensitivity in morphine-treated mice. The same dose induced a decrease in mechanical thresholds in mice that were not exposed to morphine. Values are expressed as mean %±SEM from the baseline, set to 100; n=7, *p<0.05, two-way ANOVA followed by Tukey's test; #p<0.05, vs baseline, paired t-test. (c) Motor functions are evaluated by measuring the latency to fall (in s) on the accelerating rotarod. Latencies are similar in all the groups. Values are expressed as mean %±SEM, n=7–9 mice per group, p>0.05, two-way ANOVA.
Figure 3
Figure 3
Morphine alters AMPAR subunit expression and phosphorylation in homogenates from the spinal cord dorsal horn, and promotes insertion of GluA4-contanining AMPAR in the postsynaptic density of dorsal horns. (a) Morphine (▪), but not saline (□) increases GluA4 expression in homogenates from the spinal cord dorsal horn. (b) Morphine induces an increase in the phosphorylation levels of GluA1 and GluA2 in dorsal horn homogenates. (c) Subcellular fractionation. A representative western blot shows enrichment of PSD-95 (postsynaptic marker) and the absence of synaptophysin-I (presynaptic marker) in postsynaptic density fractions from dorsal horns. (d) Morphine promotes the insertion of GluA4 at the postsynaptic density and (e) increases phosphorylation levels of GluA4. Quantification was performed relative to actin levels. Representative blots are shown. Values are expressed as mean %±SEM compared with the saline group, n=5–6 samples per group, *p⩽0.05, Mann–Whitney U-test.
Figure 4
Figure 4
Composition of postsynaptic GluA4-containing AMPAR is altered after morphine treatment. The increase in GluA4-containing AMPAR is located in laminae III–V of spinal cord dorsal horn. (a) Basal composition of GluA4-containing AMPARs in the postsynaptic density. Co-immunoprecipitation of GluA1, GluA2, and GluA3 with GluA4 shows that in naive animals, GluA4 is associated with GluA2 and GluA3, however, association between GluA4 and GluA1 is not detected. GluA2 exhibits weak association with GluA3. (b) The proportion of GluA4 associated with GluA2 is decreased 12 h after morphine (▪), but not saline (□) treatment, whereas the association between GluA4 and GluA3 remains unaltered. Quantification was performed relative to GluA4 levels detected in the pull down. Values are expressed as mean %±SEM compared with the saline group, n=3 samples per group, *p⩽0.05, Mann–Whitney U-test. (c) The proportion of GluA2 associated with GluA3 did not change after morphine treatment. Quantification was performed relative to GluA3 levels detected in the pull down. (d) The increase in GluA4 expression is located in dorsal horn laminae III–V, morphine (▪) saline (□), values represent IOD (means±SEM) n=12, *p<0.01, unpaired t-test. (e) Representative higher magnification image of GluA4 staining in dorsal horn laminae III–V from morphine-treated mouse. Immunostained puncta outline cell bodies in these laminae, consistent with the receptor having a postsynaptic location. Scale bars=10 μm.
Figure 5
Figure 5
Whole-cell patch-clamp recordings indicate morphine-induced insertion of GluA4-containing AMPAR in laminae III–V of spinal cord dorsal horn. (a) The spinal cord slice cartoon shows positioning of the electrodes. (b) AMPAR-mediated EPSCs show increased sensitivity to naspm in spinal cord slices from morphine-treated animals, which is reversed by infusion of GluA4 antibody. Sample traces (average of 20 trials) were recorded before and after naspm application (100 μM). Histograms show an average percentage inhibition (means±SEM) of EPSCs. (c) The effect of another Ca2+-permeable AMPAR blocker, IEM-1460, in slices from morphine-treated mice is similar to naspm. The amplitude of AMPAR-mediated EPSCs is plotted against time (left panel) and the histogram shows average percentage inhibition (right panel). Sample traces (average of 20 trials) were recorded before and after IEM-1460 application (50 μM). (d) Current–voltage relationships obtained by plotting AMPAR-EPSC amplitude as a function of holding potential (average I–V curves are shown). In saline-treated animal, I–V curves are almost linear showing very little contribution of Ca2+-permeable AMPAR. I–V curves obtained from a morphine-treated animal shows inward rectification – an indicator of Ca2+-permeable AMPAR insertion. Average RI calculated as EPSC (−60/+40 mV) is increased by morphine compared with saline. (e) I–V curve in morphine-treated animal is not affected by heat-inactivated anti-GluA4 included in the patch solution. Anti-GluA4 included in patch solution restores linearity of I–V curve from a morphine-treated animal. Average RI recorded from the slices of morphine-treated mice in the presence of heat-inactivated anti-GluA4 is similar to RI recorded without antibody. Anti-GluA4 included in the pipette completely reverses RI to the levels recorded in saline-treated mice, showing that Ca2+-permeable AMPARs inserted at the synapse after morphine consist of GluA4 subunits. *p<0.05.
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References

    1. Angst MS, Clark JD. Opioid-induced hyperalgesia: a qualitative systematic review. Anesthesiology. 2006;104:570–587. - PubMed
    1. Atianjoh FE, Yaster M, Zhao X, Takamiya K, Xia J, Gauda EB, et al. Spinal cord protein interacting with C kinase 1 is required for the maintenance of complete Freund's adjuvant-induced inflammatory pain but not for incision-induced post-operative pain. Pain. 2010;151:226–234. - PMC - PubMed
    1. Billa SK, Liu J, Bjorklund NL, Sinha N, Fu Y, Shinnick-Gallagher P, et al. Increased insertion of glutamate receptor 2-lacking alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors at hippocampal synapses upon repeated morphine administration. Mol Pharmacol. 2010;77:874–883. - PMC - PubMed
    1. Boehm J, Malinow R. AMPA receptor phosphorylation during synaptic plasticity. Biochem Soc Trans. 2005;33:1354–1356. - PubMed
    1. Buldakova SL, Kim KK, Tikhonov DB, Magazanik LG. Selective blockade of Ca2+ permeable AMPA receptors in CA1 area of rat hippocampus. Neuroscience. 2007;144:88–99. - PubMed

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