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, 23 (11), 4775-84

Morphine Withdrawal Increases Glutamate Uptake and Surface Expression of Glutamate Transporter GLT1 at Hippocampal Synapses

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Morphine Withdrawal Increases Glutamate Uptake and Surface Expression of Glutamate Transporter GLT1 at Hippocampal Synapses

Nan-Jie Xu et al. J Neurosci.

Abstract

Opiate abuse causes adaptive changes in several processes of synaptic transmission in which the glutamatergic system appears a critical element involved in opiate tolerance and dependence, but the underlying mechanisms remain unclear. In the present study, we found that glutamate uptake in hippocampal synaptosomes was significantly increased (by 70% in chronic morphine-treated rats) during the morphine withdrawal period, likely attributable to an increase in the number of functional glutamate transporters. Immunoblot analysis showed that expression of GLT1 (glutamate transporter subtype 1) was identified to be upregulated in synaptosomes but not in total tissues, suggesting a redistribution of glutamate transporter expression. Moreover, the increase in glutamate uptake was reproduced in cultured neurons during morphine withdrawal, and the increase of uptake in neurons could be blocked by dihydrokainate, a specific inhibitor of GLT1. Cell surface biotinylation and immunoblot analysis showed that morphine withdrawal produced an increase in GLT1 expression rather than EAAC1 (excitatory amino acids carrier 1), a neuronal subtype, at the cultured neuronal cell surface, whereas no significant change was observed in that of cultured astrocytes. Electron microscopy also revealed that GLT1 expression was markedly increased in the nerve terminals of hippocampus and associated with the plasma membrane in vivo. These results suggest that GLT1 in hippocampal neurons can be induced to translocate to the nerve terminals and express on the cell surface during morphine withdrawal. The translocation of GLT1 at synapses during morphine withdrawal provides a neuronal mechanism for modulation of excitatory neurotransmission during opiate abuse.

Figures

Figure 1.
Figure 1.
Increase of glutamate uptake in hippocampal synaptosomes during withdrawal in chronicmorphine-treatedrats. The results were presented as mean±SEM of four to six rats per group, each tested by triplicate reconstitutions. *p < 0.05 and **p < 0.01 compared with the saline group. A, B, Effect of chronic morphine treatment (Mor) and saline treatment (Sal) on different brain regions. After subcutaneous injection of morphine (10 mg/kg) twice per day at 12 hr intervals for 10 d, the glutamate uptake in the synaptosomal particles of hippocampus (Hip) was increased significantly, whereas that of other regions such as prefrontal cortex (PC), cerebellum (Cer), and brainstem (BS) were decreased. C, Glutamate uptake in synaptosomal particles of hippocampus was increased time dependently after 1,5,10 dmorphine exposure.D, The glutamate uptake was increased to the highest level 12 hr after the termination of morphine treatment and then decreased gradually 24, 36, and 48 hr later. E, Reexposure of the animals to morphine (10 mg/kg) at the 12 hr time point restored the increased glutamate uptake to the normal level 1 hr later, and the increase was initiated again by additional treatment of naloxone (Nal) (1 mg/kg).
Figure 2.
Figure 2.
Saturation analysis for glutamate uptake during morphine withdrawal by incubating the synaptosomal fractions in different glutamate concentrations. A, Glutamate uptake capacity was increased in synaptosomes during morphine withdrawal. The experiment was replicated for three times, and data were presented as mean±SEM of replicate samples in total (n = 6). B, Data as shown in Eadie-Hofstee transformations indicate an increase in Vmax of glutamate uptake. Sal, Saline treatment; Mor, morphine treatment.
Figure 3.
Figure 3.
Immunoblot characterization of glutamate transporters antibodies. A, Protein samples from the hippocampus of adult rat were analyzed by immunoblot analysis using anti-GLAST (left) and anti-GLT1 (right), respectively. Both of the antibodies recognized bands at ∼66 and ∼220 kDa. Immunoreactivity was completely abolished when each antibody was preabsorbed with 90 μm of the peptide antigen (Pep), respectively. Control experiments were done with heterologous peptides (anti-GLT1 antibody preabsorbed with GLAST peptide and anti-GLAST antibody preabsorbed with GLT1 peptide), which had no effect on the immunoreactivity in hippocampal sample immunoblots. B, Immunoblot analysis showed that the anti-GLT1 antibody could not recognize EAAC1 protein. Protein samples from HEK 293 cells expressing EAAC1 protein (HEK) and hippocampus (Hip) were subjected to SDS-PAGE and immunoblotted with the antibodies of EAAC1 and GLT1, respectively. Antibody to EAAC1 recognized bands in EAAC1 cDNA-transfected HEK 293 cells as well as in rat hippocampal tissue (left), whereas GLT1 antibody only recognized the bands in rat hippocampal tissue (right). The immunoblots were representative of three independent experiments.
Figure 4.
Figure 4.
Differential regulation of glutamate transporter subtypes in hippocampus after morphine withdrawal. A, During morphine withdrawal, the synaptosomal protein levels of GLT1 (n = 6) were significantly increased in hippocampus compared with the saline control group rather than that of GLAST (n = 5) and EAAC1 (n = 5). The monomer bands at ∼66 kDa were presented for each subtype. No significant changes were observed in the expression of either syntaxin or actin in synaptosomal fractions. B, The density of glutamate transporter immunoreactivity of synaptosomal fractions of morphine-treated rats was expressed as a percentage of that of control group (mean±SEM). *p < 0.05 compared with the saline group. Sal, Saline treatment; Mor, morphine treatment.
Figure 5.
Figure 5.
Effect of morphine withdrawal on glutamate uptake in chronic morphine-treated hippocampal neurons and astrocytes. A, B, After morphine withdrawal, the increase in glutamate uptake in cultured neurons was morphine dose (A) and time (B) dependent, whereas no significant change was seen in cultured astrocytes. The glutamate uptake in the morphinetreated group was presented as percentage of that of control group. C, Glutamate uptake in cultured astrocytes could be blocked by PDC but not by DHK treatment and was not significantly altered during morphine withdrawal after 7 d morphine (10 nm) treatment. D, Glutamate uptake in cultured neurons was increased after morphine withdrawal and could be blocked by either PDC or DHK treatment. Glutamate, 10 μm. The control rates in the cultured astrocytes and neurons were 1070 ± 93 and 748 ± 48 pmol · mg -1 · min -1 protein, respectively. The data were presented as mean ± SEM of three independent experiments performed in duplicate. Sal, Saline treatment; Mor, morphine treatment.
Figure 6.
Figure 6.
Analysis of GLT1 expression in cultured astrocytes during morphine withdrawal. A, After 7 d morphine treatment, the culture medium containing morphine was withdrawn. The astrocytes were preincubated with 0.01m PBS for 1 hr and were then biotinylated. Westernblots were probed with antibodies to GLT1 and actin. The actin bands provided an index of intracellular proteins. B, Quantitation of immunoblot of GLT1 bands was pooled from three independent experiments. GLT1 immunoreactivity values were normalized to actin in the lysate fraction. The data were expressed as a percentage of the saline-treated group for each fraction (mean ± SEM). There was no significant change in total cell or cell surface GLT1 expression. Sal, Saline treatment; Mor, morphine treatment.
Figure 7.
Figure 7.
Analysis of GLT1 and EAAC1 expression in cultured neurons during morphine withdrawal. After 7 d morphine treatment, the culture medium containing morphine was withdrawn. The cultured neurons were preincubated with 0.01 m PBS for 1 hr and were then biotinylated. A, Western blots were probed with antibodies to GLT1 and actin. B, The membranes were then striped and reprobed to EAAC1 and actin. The actin bands provided an index of intracellular proteins. The immunoblot of GLT1 and EAAC1 bands was pooled from three independent experiments. Sal, Saline treatment; Mor, morphine treatment.
Figure 8.
Figure 8.
Analysis of GLT1 mRNA in hippocampal neurons during morphine withdrawal, as revealed by in situ hybridization. Dark-field micrographs showed GLT1 mRNA distribution in hippocampal CA3 regions in the saline control group (A) and in the morphine group (B). Brightfield micrographs in the insets of A and B presented the positive pyramidal neurons containing GLT1 mRNA (arrows). Asterisks in the dark field of saline group (A) and morphine group (B) indicated the region of these neurons (arrows) located. Scale bars: A, B, 100 μm; insets in A, B, 20 μm.C, The percentage of GLT1-positive neurons in the pyramidal cell layer of CA1 and CA3 or granule cell layer of DG region was quantified, respectively, and no significant change was observed. The results were pooled from the sections (n = 4–6) of three animals in each group. D, The number of silver grains contained in GLT1-positive neurons in CA3 regions was not significantly changed during morphine withdrawal (n = 124 in control group; n = 118 in morphine-treated group). Sal, Saline treatment; Mor, morphine treatment.
Figure 9.
Figure 9.
Translocation and surface expression of GLT1 at hippocampal nerve terminals after morphine withdrawal as revealed by electron microscopy. The morphological results of the CA3 region of the hippocampus were presented as representatives in A–F. A, The ultrastructure without immunolabeling of hippocampal CA3 regions, in which synapses were formed by axonal terminals (a) and dendrites (d). A process of astrocyte was indicated with an asterisk. B, A GLT1-positive astrocyte (asterisks) was present to be juxtaposed to a synapse in the CA3 region of the saline group.C, Anaxonal terminal (a) containing gold–silver particles for GLT1 labeling was seen adjacent to a GLT1-positive astrocyte process (arrowheads) in the saline group. The gold–silver particles (arrows) in the axonal terminal were in the cytoplasm.D, AGLT1-positive axonal terminal(a) made synaptic contact with an unlabeled dendrite(d) in the morphine-treated group.E, Near a GLT1-positive as trocyte(asterisks), an unlabeled axonal terminal (a) formed a synapse with a GLT1-positive dendrite (d) in the morphinetreated group. F, A hippocampal axonal terminal (a) containing GLT1 labeling was shown close to a GLT1-positive astrocyte (arrowheads). Arrow points to gold–silver particle located in the plasma membrane of a dendrite. Double arrowheads point to the labeling in the presynaptic membrane. Scalebars:(inA) A,B,D,E, 1 μm; (in C) C,F, 0.5 μm. G, Quantitative analysis showed that the number of GLT1-positive nerve terminals was increased in both CA1 and CA3 region but not in DG region. The data were pooled from three sections of different rats in each group. The total numbers of terminals counted in CA1, CA3, and DG regions: 496, 519, and 381 in the saline group; 428, 494, and 326 in the morphine group. H, The number of gold–silver particles in nerve terminals that form synapses (Terminal) as well as associated with the plasma membrane per GLT1-positive synapse(Membrane) were quantified in the saline (n=28) and morphine-treated (n=110) groups, respectively. Both particle numbers were significantly increased in the morphine-treated group compared with the control group. *p < 0.05 and **p < 0.01 compared with the saline group. Sal, Saline treatment; Mor, morphine treatment.

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