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. 2013 May 29;33(22):9451-61.
doi: 10.1523/JNEUROSCI.5730-12.2013.

Extrasynaptic Targeting of NMDA Receptors Following D1 Dopamine Receptor Activation and Cocaine Self-Administration

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Free PMC article

Extrasynaptic Targeting of NMDA Receptors Following D1 Dopamine Receptor Activation and Cocaine Self-Administration

Pavel I Ortinski et al. J Neurosci. .
Free PMC article

Abstract

We previously showed that after repeated exposure to cocaine, D1-like dopamine receptor (D1DR) stimulation reverses plastic changes of AMPA receptor-mediated signaling in the nucleus accumbens shell. However, there is little information on the impact of cocaine self-administration on D1-NMDA receptor interactions in this brain region. Here, using whole-cell patch-clamp recordings, we assessed whether cocaine self-administration alters the effects of D1DR stimulation on synaptic and extrasynaptic NMDA receptors (NMDARs). In slices from cocaine-naive rats, pretreatment with a D1DR agonist decreased synaptic NMDAR-mediated currents and increased the contribution of extrasynaptic NMDARs. In contrast, neither cocaine self-administration alone nor cocaine experience followed by D1DR stimulation had an effect on synaptic or extrasynaptic NMDAR signaling. Activation of extrasynaptic NMDARs relies on the availability of extracellular glutamate, which is regulated primarily by glutamate transporters. In cocaine-experienced animals, relative to cocaine-naive rats, administration of a glutamate reuptake blocker, DL-threo-β-benzyloxyaspartic acid, revealed increased extrasynaptic NMDAR activity and stronger baseline activity of glutamate uptake transporters. In cocaine-naive rats, the D1DR-mediated increase in extrasynaptic NMDAR signaling was independent of the activity of glutamate reuptake transporters. Together, these results indicate that cocaine experience blunts the influence of D1DRs on synaptic and extrasynaptic NMDAR signaling. Additionally, prior cocaine self-administration limits activation of the extrasynaptic NMDAR pool by increasing glutamate reuptake. These findings outline a pattern of adaptive interactions between D1DRs and NMDARs in the nucleus accumbens shell and demonstrate upregulation of extrasynaptic NMDAR signaling as a novel consequence of cocaine self-administration.

Figures

Figure 1.
Figure 1.
Blockade of glutamate reuptake reverses the D1DR stimulation-induced decrease of NMDAR-mediated eEPSCs. A, Representative NMDAR eEPSC averages at low (2×) and high (5×) stimulation intensities following D1DR agonist (SKF38393, 10 μm) pretreatment of slices from yoked saline and cocaine-experienced animals. B, An input–output relationship of NMDAR eEPSCs illustrates reduced synaptic strength following D1DR stimulation in slices from yoked saline rats (F(3,20) = 6.206, Tukey's post hoc; **p < 0.01). C, Representative NMDAR eEPSC averages for TBOA experiments. Three traces are pictured in each experimental group: the combined NMDAR plus AMPAR eEPSC (thick black line), the AMPAR eEPSC isolated by application of dl-AP5 to the same cell (thin black line), and the subtracted NMDAR eEPSC (thick gray line). The inset for the saline ACSF group shows an expanded NMDAR eEPSC trace. In insets for all other groups, this trace is overlayed with the corresponding NMDAR eEPSCs. Amplitudes of overlayed traces are normalized for ease of comparison. Inset scale bar, 100 ms. D, A D1DR agonist-induced decrease of NMDAR eEPSCs under normal recording conditions contrasts with D1DR agonist-induced increase of NMDAR eEPSCs under conditions of glutamate reuptake transporter blockade by TBOA (30 μm; t(9) = 2.38 for SKF-treated ACSF; t(15) = 2.64 for SKF-treated TBOA; n = 5–9 cells; *p < 0.05 vs respective not-treated group). E, Cocaine treatment slightly decreases the NMDAR eEPSCs recorded in normal ACSF, but significantly increases the NMDAR eEPSCs recorded in the presence of TBOA. D1DR stimulation has no effect on NMDAR-mediated currents in either recording condition. The data are expressed as percentage difference from the respective not-treated saline group (t(17) = 2.22 for not-treated TBOA; t(11) = 2.41 for SKF-treated TBOA; *p < 0.05; n = 5–11 cells).
Figure 2.
Figure 2.
Extrasynaptic NMDAR-mediated currents increase following D1DR stimulation. A, Time course of whole-cell NMDA (100 μm, solid black line) responses after blockade of synaptic NMDARs by MK-801 (40 μm) is expressed as a fraction of whole-cell NMDA responses measured before MK-801 application. B, Bar histograms illustrating an increase in extrasynaptic NMDAR-mediated currents in slices from cocaine-naive rats following D1DR stimulation (t(11) = 2.54; *p < 0.05 vs saline no SKF; n = 6–11 cells). C, D, Current responses to the first (P1), 20th (P20) and 40th (P40) stimulation pulses illustrate use-dependent block of NMDA eEPSCs by MK-801 across the experimental groups. Current amplitudes are normalized to the first eEPSC in the saline no-SKF group for ease of comparison. E, A summary of the time course of synaptic responses in the presence of MK-801 illustrates a slight increase in the rate of MK-801 block in the saline group. F, Bar histograms summarizing the rate of block (expressed as stimulation pulse number) by MK-801.
Figure 3.
Figure 3.
Glutamate reuptake is elevated following cocaine self-administration. A, Current traces, illustrating a shift of baseline holding current during TBOA (30 μm) application (solid black lines). Dotted lines are centered at the mean holding current before application of TBOA. TBOA often induced large synchronous sEPSCs, which were clipped in this panel for display purposes. B, A summary of TBOA-induced mean current amplitudes. A significant increase is observed in slices from the cocaine-experienced group. D1DR stimulation has no effect on this increase (t(12) = 2.28 for cocaine no SKF; t(13) = 2.82 for cocaine SKF; *p < 0.05 vs respective saline group; n = 7–13 cells).
Figure 4.
Figure 4.
Blockade of glutamate reuptake reveals increased tonic NMDAR-mediated currents following cocaine self-administration and D1DR stimulation. A, NMDAR-mediated tonic currents were measured as a shift of baseline holding current following application of dl-AP5 (solid black lines) in the absence (top row) and presence (bottom row) of SKF38393 pretreatment. Dotted lines are centered at the mean holding current before application of dl-AP5. To the left of the traces are Gaussian-fitted distributions of the amplitude of baseline noise before (black) and after (gray) dl-AP5 application. The displayed fits are to the portions of the distributions not skewed by synaptic events. Notice the differences in the onset of dl-AP5 block likely due to variation in cell accessibility between slices. B, Bar histograms summarizing tonic NMDAR-mediated current amplitudes across the experimental groups (t(16) = 2.35 for saline SKF; t(17) = 2.15 for cocaine no SKF; t(15) = 2.21 for cocaine SKF; *p < 0.05 vs saline no SKF; n = 8–11 cells).
Figure 5.
Figure 5.
Blockade of glutamate reuptake reveals increased synaptic NMDA current duration following cocaine self-administration and D1DR stimulation. A, Left, NMDAR-mediated sEPSCs before (gray traces) and following (black traces) pretreatment of slices from saline controls with SKF38393. Right, The same traces are normalized to the peak amplitude to illustrate differences in the sEPSC decay time. B, Same as in A but for slices from cocaine-experienced rats. C, Bar histograms of mean NMDAR-mediated sEPSC amplitudes across the experimental groups. D, Bar histograms summarizing differences in synaptic current duration (expressed as decay time from 90 to 10% of the current peak). Again, notice that SKF38393 pretreatment is without effect in slices from cocaine-experienced rats (t(11) = 2.68 for saline no SKF; t(12) = 2.3 for cocaine no SKF; t(12) = 2.23 for cocaine SKF; *p < 0.05 vs nontreated saline; n = 6–8 cells).
Figure 6.
Figure 6.
Increase in NR2B protein following D1DR stimulation and cocaine self-administration does not correspond to an increased contribution of NR2B to whole-cell NMDAR currents. A, Quantitative Western blot analyses of NMDAR subunit protein levels. Protein levels were examined in nucleus accumbens shell homogenates from five animals in each group (*p < 0.05 vs nontreated saline, two-way ANOVA). B, Effects of NR2B antagonist, Ro 25-6981 (0.05, 0.5, 5, 50 μm), on currents evoked by whole-cell application of NMDA (100 μm). For each neuron, the responses were normalized to currents evoked by NMDA in the absence of Ro 25-6981. Notice large variability in responses. There were no significant differences between groups (n = 4–9 cells at each concentration).
Figure 7.
Figure 7.
Glutamate reuptake masks increased contribution of extrasynaptic NMDARs in slices from cocaine-experienced animals. A, Sample traces of tonic NMDAR-mediated currents recorded in the absence of glutamate reuptake blockade. Solid black lines indicate application of dl-AP5 (50 μm). B, Bar histograms summarizing tonic NMDAR-mediated current amplitudes across the experimental groups (t(11) = 3.2; **p < 0.01 vs saline no SKF; n = 6–7 cells).
Figure 8.
Figure 8.
Expression of glutamate reuptake transporters. Quantitative Western blot analyses of protein levels of glutamate reuptake transporters (GluT), GLAST, GLT-1, and EAAC-1. Protein levels were examined in nucleus accumbens shell homogenates from five animals in each group. *p < 0.05, **p < 0.01 vs nontreated saline, two-way ANOVA.
Figure 9.
Figure 9.
Chronic, but not acute, D1DR stimulation decreases NMDAR-mediated currents. A, B, Time course of whole-cell responses to NMDA (100 μm) before and after pretreatment with SKF38393 in saline controls and cocaine-experienced animals. C, Summary histograms of whole-cell NMDA responses expressed as current density (t(32) = 2.1; *p < 0.05 vs nontreated saline; n = 17–25 cells). D, A summary of eEPSCs (Evoked) and whole-cell NMDAR responses during acute application of SKF38393. In the majority of cells, NMDAR-mediated currents are potentiated during SKF38393 application.
Figure 10.
Figure 10.
Cocaine experience and D1DR stimulation alter the balance between synaptic and extrasynaptic NMDAR-mediated signaling. A working model summarizing changes in relative contributions of synaptic (black) and extrasynaptic (gray) NMDARs. Following cocaine experience, an increased contribution of extrasynaptic NMDAR receptors is masked by elevated glutamate reuptake. D1DR stimulation has no effect on NMDAR-mediated currents in this group. In saline controls, D1DR stimulation is sufficient to induce an increase in extrasynaptic NMDAR-mediated signaling. This is accompanied by a reduction of synaptic NMDAR signaling and occurs in the absence of changes in glutamate reuptake. Note that for the purposes of this diagram, glutamate reuptake occurs at unspecified (either glial or neuronal) sites.

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