Altered dopamine transporter function and phosphorylation following chronic cocaine self-administration and extinction in rats

Abstract

Cocaine binds with the dopamine transporter (DAT), an effect that has been extensively implicated in its reinforcing effects. However, persisting adaptations in DAT regulation after cocaine self-administration have not been extensively investigated. Here, we determined the changes in molecular mechanisms of DAT regulation in the caudate-putamen (CPu) and nucleus accumbens (NAcc) of rats with a history of cocaine self-administration, followed by 3weeks of withdrawal under extinction conditions (i.e., no cocaine available). DA uptake was significantly higher in the CPu of cocaine-experienced animals as compared to saline-yoked controls. DAT V(max) was elevated in the CPu without changes in apparent affinity for DA. In spite of elevated CPu DAT activity, total and surface DAT density and DAT-PP2Ac (protein phosphatase 2A catalytic subunit) interaction remained unaltered, although p-Ser- DAT phosphorylation was elevated. In contrast to the CPu, there were no differences between cocaine and saline rats in the levels of DA uptake, DAT V(max) and K(m) values, total and surface DAT, p-Ser-DAT phosphorylation, or DAT-PP2Ac interactions in the NAcc. These results show that chronic cocaine self-administration leads to lasting, regionally specific alterations in striatal DA uptake and DAT-Ser phosphorylation. Such changes may be related to habitual patterns of cocaine-seeking observed during relapse.

Figure 1. Lever responding and DA transport

A. Left panel in A: Responses on the active and inactive levers exhibited by rats averaged for the last three days of cocaine self-administration or yoked saline infusions (N=30/group). Right panel in A: Responses on the active (large symbols) and inactive (small symbols) levers exhibited by rats with a history of cocaine self-administration or yoked saline infusions on the first (day 1) and last (day 21) extinction session. Significant differences are indicated between cocaine and saline groups (*p < 0.05). B. DA transport in synaptosomes from CPu and NAcc following 3 weeks of extinction after chronic cocaine self-administration. Synaptosomal DA uptake was measured as described in “Materials and Methods”. The results are expressed as percentage of saline and data represent mean ± SEM of three experiments. *p <0.001 compared with saline (CPu) by a two-tailed Student’s t-test.

Figure 2. DA uptake kinetics, DAT-sub-cellular distribution, DAT Ser-phosphorylation and DAT-PP2 interactions in synaptosomes from CPU and NAc 3 following 3 weeks of extinction after chronic cocaine self-administration

DA uptake was measured over a range of 0.01–2.0 μM DA using CPu (A) or NAc (B) synaptosomes as described in “Materials and Methods”. Nonlinear curve fits of data for uptake used the generalized Michaelis-Menten equation. Insets show Eadie-Hofstee plots of transformation of the data. Measurements of surface DAT (C), DAT-Ser phosphorylation (D) and DAT-PP2Ac association (E) were performed as described in “Materials and Methods”. Representative immunoblots are shown in C, D and E. Averaged protein band densities from three separate experiments are shown under the immunoblots (each experiment used CPu or NAcc tissue pooled from 3 or 4 rats). Data are presented as mean ± S.E.M percent change from the saline group. *p <0.02 compared with saline (CPu) by a two-tailed Student’s t-test.