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Central amygdala GluA1 facilitates associative learning of opioid reward - PubMed

  • ️Tue Jan 01 2013

Central amygdala GluA1 facilitates associative learning of opioid reward

You-Qing Cai et al. J Neurosci. 2013.

Abstract

GluA1 subunits of AMPA glutamate receptors are implicated in the synaptic plasticity induced by drugs of abuse for behaviors of drug addiction, but GluA1 roles in emotional learning and memories of drug reward in the development of drug addiction remain unclear. In this study of the central nucleus of the amygdala (CeA), which is critical in emotional learning of drug reward, we investigated how adaptive changes in the expression of GluA1 subunits affected the learning process of opioid-induced context-reward association (associative learning) for the acquisition of reward-related behavior. In CeA neurons, we found that CeA GluA1 expression was significantly increased 2 h after conditioning treatment with morphine, but not 24 h after the conditioning when the behavior of conditioned place reference (CPP) was fully established in rats. Adenoviral overexpression of GluA1 subunits in CeA accelerated associative learning, as shown by reduced minimum time of morphine conditioning required for CPP acquisition and by facilitated CPP extinction through extinction training with no morphine involved. Adenoviral shRNA-mediated downregulation of CeA GluA1 produced opposite effects, inhibiting the processes of both CPP acquisition and CPP extinction. Adenoviral knockdown of CeA GluA2 subunits facilitated CPP acquisition, but did not alter CPP extinction. Whole-cell recording revealed enhanced electrophysiological properties of postsynaptic GluA2-lacking AMPA receptors in adenoviral GluA1-infected CeA neurons. These results suggest that increased GluA1 expression of CeA AMPA receptors facilitates the associative learning of context-drug reward, an important process in both development and relapse of drug-seeking behaviors in drug addiction.

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Conflict of interest statement

The authors declare no financial conflict of interest.

Figures

Figure 1.
Figure 1.

Morphine conditioning induces behavior of conditioned place preference (CPP) without altering protein expression of AMPA receptor GluA1 and GluA2 subunits in the central nucleus of the amygdala (CeA). A, Timeline of experimental protocol for morphine conditioning and CPP tests. Hab, habituation; S, saline; M, morphine. B, CPP in rats induced by conditioning with morphine (10 mg/kg, s.c.) paring with the non-preferred chamber after 2 (CPP test 1) or 4 (CPP test 2) conditioning sessions (n = 6 rats each group). C, CPP induced by morphine (10 mg/kg, s.c.) paring with the preferred chamber after 4 conditioning sessions (n = 6 rats/group). D,E, Western blotting (D) and summarized results (E) of GluA1 and GluA2 protein levels in synaptosomal preparations of CeA from rats (n = 5/group) conditioned with saline or morphine with established CPP after 2 or 4 conditioning sessions. GluR protein levels were normalized to that of β-actin. **p < 0.01.

Figure 2.
Figure 2.

Morphine conditioning transiently increases the protein level of synaptosomal GluA1 in CeA. A, Timeline of experimental protocol (upper) and CPP induced by one conditioning session with morphine (10 mg/kg, s.c., lower) (n = 6 rats/group). B,C, Western blot data of CeA synaptosomal GluA1 and GluA2 proteins from rats conditioned with one session of saline (S-cond.) or morphine (M-cond., 10 mg/kg) (B) or a sub-threshold morphine dose at 0.5 mg/kg (C), or injected in home cage without conditioning with the same two doses of morphine (M-inj., B,C). CeA tissues were collected 2 h after morphine conditioning as shown in A (n = 6/group). *p < 0.05, **p < 0.01.

Figure 3.
Figure 3.

Adeno-associated virus (AAV) vector-mediated overexpression of GluA1 subunits in CeA. A, Validation of AAV plasmid carrying the cDNA encoding rat GluA1(flip) in cultured CHO cells. Control cells were transfected with AAV-GFP plasmid and samples from CeA tissues were shown as positive control. B,C, Western blot data (B) and summarized results (C) of GluA1, phosphorylated GluA1 at S845 and GluA2 proteins in CeA tissues collected from rats (n = 6/group) 7 or 14 d after bilateral intra-CeA infusion of 1 μl AAV-GluA1 virus or AAV-GFP virus. D, Representative micrographs of immunostaining and illustration after CeA microinjection, showing AAV-mediated eGFP expression within the defined boundary of CeA in low magnification (a, scale bar = 500 μ

m

) and high magnification (b, scale bar = 50 μ

m

), a diagram depicting successful injection sites (black dots) within the CeA (c), and CeA GluA1 expression from a rat in an uninjected side (d, scale bar = 50 μ

m

) and in the other side injected with AAV-GluA1 in low (e, scale bar = 50 μ

m

) and high magnification (f, scale bar = 20 μ

m

). CeA preparations were prepared at post-injection day 10. **p < 0.01.

Figure 4.
Figure 4.

Overexpression of CeA GluA1 subunits facilitates associative learning of morphine reward. A, Behaviors of CPP induced by multiple conditioning sessions with saline or a low dose of morphine (0.5 mg/kg, s.c.) (n = 6 rats/group). CPP tests were conducted after each number of sessions and after the completion of all conditioning sessions (postcond., postconditioning). *p < 0.05 (compared to the pretest in the same group). B, CPP behaviors after similar conditioning sessions with saline (n = 6) or morphine (0.5 mg/kg) in rats injected into CeA with AAV-GluA1 (n = 9) or AAV-GFP (n = 6). *p < 0.05 (compared to the pretest in the same group); #p < 0.05 (compared to the Morphine/AAV-GFP group). C, CPP behaviors in CeA AAV-GluA1- or AAV-GFP-injected rats conditioned with multiple sessions of morphine at 3 mg/kg, followed by daily conditioning sessions of extinction training with saline only (n = 9 rats/group). *p < 0.05 (compared to the pretest in the same group); #p < 0.05 (compared to the AAV-GFP group); **p < 0.01; n.s., not significant. D, CPP behaviors in normal rats (n = 8/group) induced by conditioning with morphine at 3 mg/kg and 10 mg/kg, followed by daily sessions of extinction training. *p < 0.05 (compared to the pretest in the same group); #p < 0.05 (compared to the 3 mg/kg group).

Figure 5.
Figure 5.

AAV-GluA1-shRNA-mediated downregulation of CeA GluA1 subunits inhibits associative learning of morphine reward. A, Western blotting analysis of CeA synaptosomal GluA1 (GA1) and GluA2 proteins in rats (n = 6/group) with bilateral CeA injection of AAV-GluA1-shRNA or AAV-scrambled (Scr) shRNA. CeA tissues were collected at post-injection day 10. **p < 0.01. B, Representative micrographs of GluA1 immunoreactivity and GFP expression in CeA neurons from a rat after CeA injection of AAV-scrambled shRNA (a–c) or AAV-GluA1-shRNA (d–f). Scale bars, 50 μ

m

. C, CPP behaviors in rats with bilateral CeA injection of AAV-scrambled shRNA or AAV-GluA1-shRNA after multiple conditioning sessions with saline (n = 6 rats/group) or morphine (3 mg/kg, n = 10 rats/group), followed by daily conditioning sessions of extinction training. *p < 0.05 (compared to the pretest in the same group); #p < 0.05 (compared to the morphine/scrambled shRNA group).

Figure 6.
Figure 6.

GluA1 overexpression increases synaptic properties of GluA2-lacking AMPA receptors in CeA neurons. A, Illustration of stimulation (S) placement in BLA, and recording (R) of a viral vector-transfected neuron and EPSC in a CeA slice. A transfected cell was identified by the GFP fluorescence (top middle) and recorded in whole-cell configuration (top right) with a typical postsynaptic current (PSC) and excitatory postsynaptic current (EPSC) in the presence of picrotoxin (50 μ

m

) (bottom right). B, Representative AMPA EPSCs and NMDA EPSCs in a control cell and in transfected cells from animals after single-side CeA injection of the mixture (GluA1-GFP) of AAV-GluA1 and AAV-GFP vectors (3:1) or AAV-GluA1-shRNA-GFP vector. C, Summarized results of the AMPA/NMDA EPSC ratio in the three indicated cell groups. Numbers in columns are cell numbers in each group. D, EPSCs at holding potentials of +40 mV and −70 mV in the presence of the NMDA receptor antagonist

d

-AP5 (50 μ

m

) (top) and the calculated rectification index of EPSCs (bottom) in the three cell groups. E, EPSCs (top) and summarized results (bottom) before and during application of the GluA2-lacking AMPA receptor antagonist NASPM (100 μ

m

) in the indicated cell groups. F, Evoked EPSC pairs (50 ms apart, top) and summarized data of paired-pulse ratios (bottom) in the three cell groups. *p < 0.05, **p < 0.01.

Figure 7.
Figure 7.

GluA2-lacking AMPA receptors in CeA are involved in acquired behavior of opioid reward. A, Behaviors of CPP in rats conditioned in 4 sessions with PBS as vehicle, morphine (10 mg/kg) without or with bilateral pre-CeA infusion (15 min before) of NASPM (40 μg, each side), or vehicle with CeA pre-infusion of NASPM. **p < 0.01 (compared to the pretest); ##p < 0.01 (compared between the indicated two groups) (n = 8 rats in each group). B, CPP behaviors in similar groups of rats with morphine substituted by AMPA (100 ng, each side) infused similarly into CeA in the conditioning procedure. **p < 0.01 (compared to the pretest); ##p < 0.01 (compared between the indicated two groups); n = 8 rats in each group.

Figure 8.
Figure 8.

Downregulation of CeA GluA2 facilitates CPP but does not change CPP extinction. A, Representative Western blots (top) and summarized data (bottom) of synaptosomal and cytoplasmic GluA2 and GluA1 proteins in the CeA from rats (n = 4/group) treated with CeA infusion of AAV-scrambled (scr) shRNA or AAV-GluA2-shRNA. B, CPP behaviors in rats with bilateral CeA injection of AAV-scrambled shRNA (n = 8) or AAV-GluA2-shRNA (n = 10) after multiple conditioning sessions with morphine (3 mg/kg, s.c.), followed by daily conditioning sessions of extinction training with saline only. *p < 0.05 (compared to the pretest in the same group); #p < 0.05 (compared to the scrambled shRNA group). C,D, Exploration times (C) and novel preference (D) in the novel object recognition test in rats (n = 6 each group) after CeA infusion of AAV-GFP, AAV-GluA1, or AAV-GluA1-shRNA vectors.

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