Extinction of morphine-dependent conditioned behavior is associated with increased phosphorylation of the GluR1 subunit of AMPA receptors at hippocampal synapses

Abstract

In abstinent opiate addicts, relapse can be triggered by exposure to environmental cues associated with drug use; thus, the disruption of these learned associations may be an effective approach for reducing relapse. Interestingly, glutamatergic systems are thought to be involved in opiate-induced behavioral plasticity. In this study, changes in expression and phosphorylation levels of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor subunits (GluR1, GluR2) in the hippocampus were investigated in rats showing a conditioned response (CR) to an opiate-paired environment as well as in animals in which this conditioned behavior was extinguished. Additionally, another set of animals went through a drug-unpaired paradigm (without conditioning) in order to examine the effects of the pharmacology of the drug itself. Subcellular fractionation techniques were used to analyse the local distribution of AMPA glutamate subunits within the synapse, especially at the postsynaptic density (PSD). Results showed that morphine-dependent CRs did not alter expression or redistribution of GluR1 or GluR2; however, the unpaired administration of morphine resulted in an increase in the phosphorylation of the GluR1 subunit at extrasynaptic sites. Interestingly, the extinction of the CR significantly increased phosphorylation of the GluR1 subunit at the PSD. Therefore we propose that, within the synapse, the phosphorylation of the GluR1 subunit at the PSD may be a key mechanism in the extinction of opiate-associated CRs.

Figure 1

Schematic representation of the experimental design. (A, B) After a single preconditioning test (day 0), rats received two daily conditioning sessions from days 1 to 4. Morphine-conditioned animals received morphine (10 mg/kg, s.c) in one side of the CPP chamber and saline in the other side. Saline controls received saline in both chambers. Expression test was performed on day 5. (A) Upon expression test, half of the animals (morphine-CPP and saline-CPP, n=8/subgroup) were immediately sacrificed and their hippocampi dissected. (B) Extinction training was performed with the remaining animals. Following expression test, animals received a single daily extinction training. When an animal reached extinction criteria it was immediately sacrificed along with a randomly chosen saline control (morphine-CPP extinction, saline-CPP extinction, n=7–8/subgroup). (C) In the unpaired group, rats received 10 mg/kg s.c, morphine or saline (morphine- and saline-unpaired respectively, n=9 per group) following the same protocol than that described for the place preference paradigm but without any conditioning. Animals were sacrificed 12 h upon last morphine administration.

Figure 2

(A) Conditioned place preference to morphine. Animals trained with morphine exhibited a significant place preference for the morphine-paired compartment. Rats were conditioned with morphine (10 mg/kg s.c.) or saline for 4 days (n=16 per group). Data represent the time spent (sec) in the morphine-paired chamber versus the saline-paired chamber for the morphine treated rats and time spent in the black chamber versus the white chamber for the saline group (mean ± SEM). ***p<0.001, two-tailed paired t-test. (B) Extinction of conditioned placed preference to morphine. Following initial CPP testing, rats were given daily tests until the extinction criteria was reached. Place preference for the morphine paired chambers gradually diminished over time. Summary of the variability between subjects in the extinction of morphine-dependent CPP. Time to extinction ranged from 3 to 13 days depending on the animal.

Figure 3

Biochemical validation of the subcellular fractionation protocol performed to isolate PSD from rat hippocampus. Enrichment of PSD proteins during fractionation was confirmed by western blotting. (A) 10 ug of protein from hippocampus homogenate (Homo), synaptosomes (Syn), synaptic junctions (Syn Junc), PSD, and presynaptic fraction (PAZ) were separated by SDS-PAGE and probed with antibodies to known postsynaptic (PSD95, CaMKII) and presynaptic markers (syntaxin 1). (B) Synaptic localization of AMPA receptor subunits within the hippocampal synapse. Western blot analysis indicated that phosphorylation of the GluR1 subunit and expression of GluR1 and GluR2 subunits of AMPA receptors is increased at the PSD and absent at the presynaptic fraction (PAZ).

Figure 4

The expression of morphine-CPP does not induce changes in expression of GluR1 and GluR2 receptors in synaptic fractions. Upon initial CPP testing, rats were sacrificed, hippocampi dissected, and subcellular fractionation performed in individual samples. Expression and phosphorylation of AMPA subunits within the synapse were analyzed by western blotting using antibodies to phospho-GluR1 Ser845 (pGluR1) (A), GluR1 (B) or GluR2 (C). Although rats exhibited a strong CPP to morphine, western blotting analysis did not show any changes in the phosphorylation state or the expression levels of GluR1 (A, B) and GluR2 (C)subunits of AMPA receptors in any synaptic fraction studied. A representative western blot is shown for each of the receptor subunits studied. Measurement of pGluR1, GluR1 and GluR2 levels were normalized to actin levels in the lane. Data (mean ± SEM) are normalized to saline controls and represent the sum of 8 individual animals. Homog, homogenate; Synap, synaptosomal fraction; S, saline; M, morphine.

Figure 5

The extinction of morphine-CPP increases phosphorylation of GluR1 at the PSD. Upon initial CPP testing, animals underwent extinction training. When an animal reached extinction criteria it was sacrificed along with a randomly chosen saline control. Subcellular fractionation was performed in individual samples. Expression and phosphorylation of AMPA subunits within the synapse were analyzed by western blotting using antibodies to phospho-GluR1 Ser845 (pGluR1) (A), GluR1 (B) or GluR2 (C). (A) Extinction of morphine-CPP led to a significant increase in the phosphorylation of GluR1 at the PSD; no significant changes were observed in other synaptic fractions. (B, C) No changes were observed in the levels of GluR1 and GluR2 in any of the synaptic fractions studied. A representative western blot is shown for each of the receptor subunits studied. Measurement of pGluR1, GluR1 and GluR2 levels were normalized to actin levels in the lane. Data (mean ± SEM) are normalized to saline controls and represent the sum of 7–8 individual animals. *p<0.05, two-tailed paired t-test. Homog, homogenate; Synap, synaptosomal fraction; S, saline; M, morphine.

Figure 6

Repeated unpaired administration of morphine (without any conditioning) leads to changes in the phosphorylation and expression of GluR1 and GluR2 receptors in synaptic fractions. Animals were sacrificed 12 h upon the last morphine administration (10 mg/kg s.c.) and their hippocampi dissected. Subcellular fractionation was performed in individual samples. Expression and phosphorylation of AMPA subunits within the synapse were analyzed by western blotting using antibodies to phospho-GluR1 Ser845 (pGluR1) (A), GluR1 (B) or GluR2 (C). (A) Unpaired morphine administration led to a significant increase in the phosphorylation of GluR1 at the synaptosomal fraction with no changes in other synaptic fractions. (B, C) Levels of GluR1 and GluR2 were decreased locally at the PSD. A representative western blot is shown for each of the receptor subunits studied. Measurement of pGluR1, GluR1 and GluR2 levels were normalized to actin levels in the lane. Data (mean ± SEM) are normalized to saline controls and represent the sum of 8 individual animals. *p<0.05; **p<0.01, two-tailed paired t-test. Homog, homogenate; Synap, synaptosomal fraction; S, saline; M, morphine.