Upregulation of nerve growth factor in central amygdala increases sensitivity to opioid reward

Abstract

The rewarding properties of opioids are essential driving force for compulsive drug-seeking and drug-taking behaviors in the development of opioid-mediated drug addiction. Prior drug use enhances sensitivity to the rewarding effects of subsequently used drugs, increasing vulnerability to relapse. The molecular mechanisms underlying this reward sensitization are still unclear. We report here that morphine that induced reward sensitization, as demonstrated by reinstatement of the behavior of conditioned place preference (CPP) with sub-threshold priming morphine, epigenetically upregulated the output activity of Ngf encoding the nerve growth factor (NGF) by increasing histone H4 acetylation in the rat central nucleus of the amygdala (CeA). NGF locally infused into the CeA mimicked the morphine effect in inducing new functional delta-opioid receptor (DOR) that was required for the reward sensitization, and morphine-induced reward sensitization was inhibited by blocking NGF receptor signaling in the CeA. Histone deacetylase inhibitors that increased the acetylation level at the Ngf promoter and NGF expression in the CeA also induced reward sensitization in a CeA NGF signaling- and DOR-dependent manner. Furthermore, CeA-applied NGF substituted prior morphine to induce reward sensitization in naive rats and also substituted priming morphine to reinstate the CPP induced by prior morphine. Thus, epigenetic upregulation of NGF activity in the CeA may promote the behavior of opioid reward and increase the sensitivity to the rewarding effect of subsequent opioids, a potentially important mechanism in drug addiction.

Figure 1

Morphine epigenetically activates the Ngf gene and the expression of nerve growth factor (NGF) in the central nucleus of the amygdala (CeA) of rats with increased sensitivity to morphine reward. (a) Behaviors of conditioned place preference (CPP), expressed as the time spent in the conditioning chamber before (pretest) and after conditioning (posttest), in saline-conditioned rats (one-way ANOVA (same below): F_(1,12)=0.009, n=13) and in morphine-conditioned rats (F_(1,25)=58.47, n=26). (b) CPP behaviors in morphine-conditioned rats after CPP establishment (F_(1,9)=26.59), extinction (F_(1,9)=52.98), and reinstatement (F_(1,9)=42.06) by a single priming dose of morphine (1 mg/kg, i.p.), and in saline-conditioned naive rats either as control or after conditioning with the priming morphine (F_(1,6)=1.08). N=7–15 rats in each group. (c) Normalized levels of global acetylated histone H4, expressed as the ratio of DNA associated with the acetylated H4 to input DNA, in CeA tissues from saline-conditioned naive rats, from morphine-conditioned rats with CPP established, extinguished and reinstated, and from saline-conditioned naive rats after conditioning with priming morphine. (d, e) Levels of H4 acetylation across the Ngf promoter region (d) and across the Gapdh promoter region (e) in similar groups of rats to those in (a). N=5–7 rats for each group. (f) Changes in NGF mRNA levels in CeA tissues from similar groups of morphine-conditioned rats at the three indicated CPP stages and of saline-conditioned rats without or after conditioning with priming morphine. N=5 rats for each group. (g, h) Representative western blots (g) and group data of NGF protein levels normalized to β-actin (h) in CeA tissues from similar rat groups as indicated. N=5 rats for each group. Summarized data are mean±SEM. ^*p<0.05, ^**p<0.01. Establ., establishment; Reinst., reinstatement.

Figure 2

Morphine induces functional DOR that is required for morphine-induced reward sensitization. (a) Representative glutamatergic excitatory postsynaptic currents (EPSCs) in control and in the presence of [D-Pen²,D-Pen⁵]-enkephalin (DPDPE, 1 μM), a selective agonist of the delta-opioid receptor (DOR), in a CeA neuron from a saline-conditioned rat, from morphine-conditioned rats with CPP established, extinguished, and reinstated, and from a saline-conditioned rat after conditioning with priming morphine. (b) Summarized data of the DPDPE effects normalized to controls in the same groups of rats as in (a). N=9–20 cells for each group. (c) Effect of the DOR antagonist naltrindole (NTD), infused bilaterally into the CeA (0.45 μg in 0.5 μl each side) 1 day after posttest, on baseline preference in saline-conditioned naive rats (F_(1,5)=1.32, n=5) and in morphine-conditioned rats with CPP (F_(1,6)=20.90, n=7 rats). For morphine vs saline comparison, F_(1,6)=18.68. (d) Effect of NTD, infused similarly into the CeA 1 day after CPP reinstatement, on the priming morphine-reinstated CPP (F_(1,8)=19.28, n=9 rats). Establishment: F_(1,8)=22.47, extinction: F_(1,8)=49.62, and reinstatement: F_(1,8)=33.16. Summarized data are mean±SEM. ^**p<0.01.

Figure 3

NGF signaling in the CeA is necessary for morphine-induced reward sensitization. (a) Representative effect of DPDPE on EPSCs from a group of CeA neurons (control, 320±22 pA, DPDPE, 224±17 pA, n=9 out of 12 (75%) cells, p<0.01) in naive slices treated with NGF (100 ng/ml) in vitro for 4 h. (b) DPDPE effect on EPSCs (control, 233±43 pA, DPDPE, 148±29 pA, n=6 out of 8 (75%) cells, p<0.01) in similar NGF-treated slices from morphine-conditioned rats with CPP extinction. (c, d) DPDPE effects on EPSCs in CeA slices treated in vitro with the tyrosine receptor kinase (Trk) inhibitor K252a (0.2 μM) from rats with morphine-established CPP (c, control, 240±29 pA, DPDPE, 232±28 pA, n=8, p>0.05) or with priming morphine-reinstated CPP (d, control, 228±21 pA, DPDPE, 214±21 pA, n=7, p>0.05). (e, f) Effects of pre-treatment with bilateral CeA infusion of K252a (24 ng) on preference behaviors in morphine- (F_(1,5)=0.65) or saline- (F_(1,3)=0.06) conditioned rats (e) and in saline- or morphine-conditioned rats after CPP extinction (F_(1,5)=0.42) (f). Establishment: F_(1,5)=10.41 and extinction: F_(1,5)=16.71. N=4–6 rats for each group. Summarized data are mean±SEM. ^**p<0.01.

Figure 4

Histone hyperacetylation induces reward sensitization through CeA NGF signaling. (a) Preference behaviors before (pretest) and after (posttest) conditioning with priming morphine (1 mg/kg, i.p.) in naive rats pre-treated 4 h before with bilateral CeA infusion of vehicle (n=10 rats) or the histone deacetylase inhibitor Trichostatin A (TsA, 2.5 μg, F_(3,40)=24.82, n=12 rats). (b) Normalized levels of global acetylated histone H4, acetylated H4 across the Ngf promoter region and NGF mRNA in CeA tissues from priming morphine-conditioned rats pre-treated by CeA infusion of vehicle or TsA as in (a). N=4–5 rats for each group. (c–e) Western blot lanes (c, d) and summarized data (e) of NGF protein normalized to β-actin and synaptosomal DOR protein normalized to synaptophysin in CeA tissues from rat groups similar to those in (a). (f, g) Preference behaviors in priming morphine-conditioned rats pre-treated with CeA infusion of vehicle and TsA plus the Trk antagonist K252a (24 ng, F_(3,14)=24.52, f), or TsA plus the DOR antagonist NTD (0.45 μg, F_(3,14)=5.82, g). (h) Western blots and summarized data of synaptosomal DOR protein in CeA tissues from the two rat groups as in (f). Summarized data are mean±SEM. ^*p<0.05, ^**p<0.01. Syn, synaptosomal; Synphsn, synaptophysin; Veh, vehicle.

Figure 5

NGF substitutes morphine to induce reward sensitization. (a) Preference behaviors in priming morphine-conditioned naive rats pre-treated by CeA infusion of saline (F_(1,6)=1.08, n=7 rats) or NGF (0.05 μg, F_(1,5)=14.80, n=6 rats) or NGF plus NTD (0.45 μg, F_(1,5)=34.25, n=6 rats), and in saline-conditioned rats (n=4) with similar CeA infusion of saline or NGF (F_(1,3)=0.08). (b) Conditioning with NGF, infused bilaterally into the CeA (0.05 μg), reinstated prior morphine-induced CPP. After the extinction of morphine-induced CPP (saline/morphine, F_(5,25)=9.89; NGF/morphine, F_(5,35)=10.86), rats were conditioned with NGF in NGF group (n=8) or saline in saline group (n=6) and then tested on the 3 days (D1–D3) following the completion of conditioning (two-way ANOVA: conditioning session, F_(5,60)=25.21, p<0.001; interaction: F_(5,60)=7.63, p<0.001). (c) A photomicrograph showing the infusion site in the CeA marked by an injected dye (arrow). Scale bar is 1 mm. Summarized data are mean±SEM. ^*p<0.05, ^**p<0.01, ^***p<0.001. Mor, morphine.